Oligomeric phosphite, phosphodiester, Phosphorothioate and phosphorodithioate compounds and intermediates for preparing same

ABSTRACT

Synthetic processes are provided wherein oligomeric compounds are prepared having phosphodiester, phosphorothioate, and phosphorodithioate covalent linkages. Also provided are synthetic intermediates useful in such processes.

FIELD OF THE INVENTION

This invention relates to methods for the preparation of oligomericcompounds having phosphite, phosphodiester, phosphorothioate, orphosphorodithioate linkages, and to intermediates useful in theirpreparation.

BACKGROUND OF THE INVENTION

Oligonucleotides and their analogs have been developed and used inmolecular biology in certain procedures as probes, primers, linkers,adapters, and gene fragments. Modifications to oligonucleotides used inthese procedures include labeling with nonisotopic labels, e.g.fluorescein, biotin, digoxigenin, alkaline phosphatase, or otherreporter molecules. Other modifications have been made to the ribosephosphate backbone to increase the nuclease stability of the resultinganalog. These modifications include use of methyl phosphonate,phosphorothioate, and phosphorodithioate linkages, and 2'-O-methylribose sugar units. Further modifications include those made to modulateuptake and cellular distribution. With the success of these compoundsfor both diagnostic and therapeutic uses, there exists an ongoing demandfor improved oligonucleotides and their analogs.

It is well known that most of the bodily states in multicellularorganisms, including most disease states, are effected by proteins. Suchproteins, either acting directly or through their enzymatic or otherfunctions, contribute in major proportion to many diseases andregulatory functions in animals and man. For disease states, classicaltherapeutics has generally focused upon interactions with such proteinsin efforts to moderate their disease-causing or disease-potentiatingfunctions. In newer therapeutic approaches, modulation of the actualproduction of such proteins is desired. By interfering with theproduction of proteins, the maximum therapeutic effect may be obtainedwith minimal side effects. It is a general object of such therapeuticapproaches to interfere with or otherwise modulate gene expression,which would lead to undesired protein formation.

One method for inhibiting specific gene expression is with the use ofoligonucleotides, especially oligonucleotides which are complementary toa specific target messenger RNA (mRNA) sequence. Severaloligonucleotides are currently undergoing clinical trials for such use.Phosphorothioate oligonucleotides are presently being used as antisenseagents in human clinical trials for various disease states, includinguse as antiviral agents.

Transcription factors interact with double-stranded DNA duringregulation of transcription. Oligonucleotides can serve as competitiveinhibitors of transcription factors to modulate their action. Severalrecent reports describe such interactions (see Bielinska, A., et. al.,Science, 1990, 250, 997-1000; and Wu, H., et. al., Gene, 1990, 89,203-209).

In addition to such use as both indirect and direct regulators ofproteins, oligonucleotides and their analogs also have found use indiagnostic tests. Such diagnostic tests can be performed usingbiological fluids, tissues, intact cells or isolated cellularcomponents. As with gene expression inhibition, diagnostic applicationsutilize the ability of oligonucleotides and their analogs to hybridizewith a complementary strand of nucleic acid. Hybridization is thesequence specific hydrogen bonding of oligomeric compounds viaWatson-Crick and/or Hoogsteen base pairs to RNA or DNA. The bases ofsuch base pairs are said to be complementary to one another.

Oligonucleotides and their analogs are also widely used as researchreagents. They are useful for understanding the function of many otherbiological molecules as well as in the preparation of other biologicalmolecules. For example, the use of oligonucleotides and their analogs asprimers in PCR reactions has given rise to an expanding commercialindustry. PCR has become a mainstay of commercial and researchlaboratories, and applications of PCR have multiplied. For example, PCRtechnology now finds use in the fields of forensics, paleontology,evolutionary studies and genetic counseling. Commercialization has ledto the development of kits which assist non-molecular biology-trainedpersonnel in applying PCR. Oligonucleotides and their analogs, bothnatural and synthetic, are employed as primers in such PCR technology.

Oligonucleotides and their analogs are also used in other laboratoryprocedures. Several of these uses are described in common laboratorymanuals such as Molecular Cloning, A Laboratory Manual, Second Ed., J.Sambrook, et al., Eds., Cold Spring Harbor Laboratory Press, 1989; andCurrent Protocols In Molecular Biology, F. M. Ausubel, et al., Eds.,Current Publications, 1993. Such uses include as syntheticoligonucleotide probes, in screening expression libraries withantibodies and oligomeric compounds, DNA sequencing, in vitroamplification of DNA by the polymerase chain reaction, and insite-directed mutagenesis of cloned DNA. See Book 2 of MolecularCloning, A Laboratory Manual, supra. See also "DNA-protein interactionsand The Polymerase Chain Reaction" in Vol. 2 of Current Protocols InMolecular Biology, supra.

Oligonucleotides and their analogs can be synthesized to have customizedproperties that can be tailored for desired uses. Thus a number ofchemical modifications have been introduced into oligomeric compounds toincrease their usefulness in diagnostics, as research reagents and astherapeutic entities. Such modifications include those designed toincrease binding to a target strand (i.e. increase their meltingtemperatures, Tm), to assist in identification of the oligonucleotide oran oligonucleotide-target complex, to increase cell penetration, tostabilize against nucleases and other enzymes that degrade or interferewith the structure or activity of the oligonucleotides and theiranalogs, to provide a mode of disruption (terminating event) oncesequence-specifically bound to a target, and to improve thepharmacokinetic properties of the oligonucleotide.

The chemical literature discloses numerous processes for couplingnucleosides through phosphorous-containing covalent linkages to produceoligonucleotides of defined sequence. One of the most popular processesis the phosphoramidite technique (see, e.g., Advances in the Synthesisof Oligonucleotides by the Phosphoramidite Approach, Beaucage, S. L.;Iyer, R. P., Tetrahedron, 1992, 48, 2223-2311 and references citedtherein), wherein a nucleoside or oligonucleotide having a free hydroxylgroup is reacted with a protected cyanoethyl phosphoramidite monomer inthe presence of a weak acid to form a phosphite-linked structure.Oxidation of the phosphite linkage followed by hydrolysis of thecyanoethyl group yields the desired phosphodiester or phosphorothioatelinkage.

The phosphoramidite technique, however, has significant disadvantages.For example, cyanoethyl phosphoramidite monomers are quite expensive.Although considerable quantities of monomer go unreacted in a typicalphosphoramidite coupling, unreacted monomer can be recovered, if at all,only with great difficulty.

Another disadvantage of using a β-eliminating cyanoethoxy group isformation of acrylonitrile upon removal of the phosphorus protectinggroup. Acrylonitrile is a highly toxic agent as well as a suspectedcarcinogen (See 1994-1995 Aldrich Chemical Company Catalog, at page 32).Acrylonitrile is also suspected of forming cyclic structures withthymidine resulting in oligomeric compounds having decreasedhybridization ability. These modified oligomeric compounds areundesirable because they are difficult to separate from the desiredoligomeric compound.

Consequently, there remains a need in the art for synthetic methods thatwill overcome these problems.

Several processes are known for the solid phase synthesis ofoligonucleotide compounds. These are generally disclosed in thefollowing U.S. Pat. No. 4,458,066; issued Jul. 3, 1984; U.S. Pat. No.4,500,707, issued Feb. 19, 1985; and U.S. Pat. No. 5,132,418, issuedJul. 21, 1992. Additionally, a process for the preparation ofoligonucleotides using phosphoramidite intermediates is disclosed inU.S. Pat. No. 4,973,679, issued Nov. 27, 1990.

A process for the preparation of phosphoramidites is disclosed in U.S.Pat. No. 4,415,732, issued Nov. 15, 1983.

Phosphoramidite nucleoside compounds are disclosed in U.S. Pat. No.4,668,777, issued May 26, 1987.

A process for the preparation of oligonucleotides using a β-eliminatingphosphorus protecting group is disclosed in U.S. Pat. No. Re. 34,069,issued Sep. 15, 1992.

A process for the preparation of oligonucleotides using a β-eliminatingor allylic phosphorus protecting group is disclosed in U.S. Pat. No.5,026,838, issued Jun. 25, 1991.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide methods for thepreparation of oligomeric compounds having phosphite, phosphodiester,phosphorothioate, or phosphorodithioate containing covalent linkages. Itis a further object of the present invention to provide syntheticintermediates useful in such processes. Other objects will be apparentto those skilled in the art.

These objects are satisfied by the present invention, which providesmethods for the preparation of oligomeric compounds having phosphite,phosphodiester, phosphorothioate, or phosphorodithioate containingcovalent linkages.

In one aspect of the present invention, methods are provided for thepreparation of oligomeric compounds comprising a moiety having theFormula IX: ##STR1## wherein: Z is CN, --Si(R₉)₃, halogen, NO₂, alkaryl,sulfoxyl, sulfonyl, thio, substituted sulfoxyl, substituted sulfonyl, orsubstituted thio, wherein the substituents are selected from the groupconsisting of alkyl, aryl, or alkaryl;

each R₉ is, independently, alkyl having 1 to about 10 carbon atoms, oraryl having 6 to about 10 carbon atoms;

X₁ is O or S;

comprising the steps of:

(a) providing a compound having the Formula II: ##STR2## wherein: eachR₁, is, independently, H, --OH, --F, or --O--X₃ --D;

X₃ is alkyl having from 1 to 10 carbons;

D is H, amino, protected amino, alkyl substituted amino, imidazole, or(--O--X₃)_(p), where p is 1 to about 10;

each X₂ is O or S;

R₃ and R_(3a) are each hydrogen, a hydroxyl protecting group, or alinker connected to a solid support;

each B, independently, is a naturally occurring or non-naturallyoccurring nucleobase or a protected naturally occurring or non-naturallyoccurring nucleobase;

n is 0 to about 50;

each Q is --X₁ H or --X₁ --CH₂ --CH═CH--CH₂ --Z;

R₅ is --N(R₆)₂, or a heterocycloalkyl or heterocycloalkenyl ringcontaining from 4 to 7 atoms, and having up to 3 heteroatoms selectedfrom the group consisting of nitrogen, sulfur, and oxygen;

R₆ is straight or branched chain alkyl having from 1 to 10 carbons;

(b) reacting the compound of Formula II with a compound having theFormula III: ##STR3## wherein R_(3a) is hydrogen;and R₂ is a hydroxylprotecting group, or a linker connected to a solid support, providedthat R₂ and R₃ are not both simultaneously a linker connected to a solidsupport; to form the oligomeric compound.

Some preferred embodiments of the methods of the invention furthercomprise the step of oxidizing the oligomeric compound. In somepreferred embodiments, the methods of the invention further comprisetransforming the oxidized oligomeric compound to form a further compoundhaving the Formula III, where n is increased by 1.

Preferably, the methods of the invention comprise a capping step, eitherprior to or after the oxidation step.

Other preferred embodiments of the invention further comprise the stepof cleaving the oligomeric compound to produce a compound having theFormula I: ##STR4##

In some preferred embodiments of the invention, Z is CN. In otherpreferred embodiments of the invention, each R₆ is isopropyl.

In preferred embodiments, X₁ and X₂ can each independently be O or S.

In some preferred embodiments the compound of Formula II is obtained byreaction of a compound having the Formula V: ##STR5## with a compoundhaving the Formula VI:

    (R.sub.5).sub.2 P--X.sub.1 --CH.sub.2 --CH═CH--CH.sub.2 --ZVI

in the presence of an acid. Preferably, R₅ is N,N-diisopropylamino.

Also provided in accordance with the invention are novel compoundshaving the Formula VII:

    A--X.sub.1 --CH.sub.2 --CH═CH--CH.sub.2 --Z            VII

wherein:

X₁ is O or S;

A is (R₇) (R₈)P--;

R₈ is R₅, or has the Formula X: ##STR6## wherein: each R₁, is,independently, H, --OH, --F, --O--X₃ --D;

X₃ is alkyl having from 1 to 10 carbons;

D is H, amino, protected amino, alkyl substituted amino, imidazole, or(--O--X₃)_(p), where p is 1 to about 10;

each X₂ is O or S;

R₅ is --N(R₆)₂, or a heterocycloalkyl or heterocycloalkenyl ringcontaining from 4 to 7 atoms, and having up to 3 heteroatoms selectedfrom the group consisting of nitrogen, sulfur, and oxygen;

each Q is --X₁ H or --X₁ --CH₂ --CH═CH--CH₂ --Z;

m is 0 to about 50;

each B, independently, is a naturally occurring or non-naturallyoccurring nucleobase or a protected naturally occurring or non-naturallyoccurring nucleobase; and

R₇ is R₅, or has the Formula VIII: ##STR7## wherein: R₃ is hydrogen, ahydroxyl protecting group, or a linker connected to a solid support; and

n is 0 to about 50; with the proviso that the sum of m and n do notexceed 50; and

Z is CN, --Si(R₉)₃, halogen, NO₂, alkaryl, sulfoxyl, sulfonyl, thio,substituted sulfoxyl, substituted sulfonyl, or substituted thio, whereinthe substituents are selected from the group consisting of alkyl, aryl,or alkaryl;

each R₉ is, independently, alkyl having 1 to about 10 carbon atoms, oraryl having 6 to about 10 carbon atoms;

In some preferred embodiments of the compounds of the invention Z is CN.In other preferred embodiments Z is Si(R₉)₃. In further preferredembodiments A is H or --P(R₅)₂.

In some preferred embodiments R₅ is --N(CH(CH₃)₂)₂, and other preferredembodiments R₇ has the Formula VII.

Preferably, n is 1 to 30, with 1 to about 20 being more preferred. Insome preferred embodiments n is 0.

In more preferred embodiments, Z is CN, X₁ is O, and A is H; or Z is CN;X₁ is S; and A is H. In other preferred embodiments Z is CN, X₁ is O,and each R₆ is isopropyl. In further preferred embodiments Z is CN, X₁is S, and each R₆ is isopropyl.

In particularly preferred embodiments, the compounds of the inventionhave the Formula IV: ##STR8## wherein R₂ is preferably a linkerconnected to a solid support, or hydrogen.

In preferred embodiments m and n are each 0; or Z is CN, and X₁ is O.

The present invention also provides products produced by the methods ofthe invention.

DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention provides methods for the preparation of oligomericcompounds having phosphite, phosphodiester, phosphorothioate, orphosphorodithioate linkages, and to intermediates useful in theirpreparation.

In some preferred embodiments of the invention, methods are provided forthe preparation of an oligomeric compound comprising a moiety having theFormula IX: ##STR9## wherein: Z is CN, --Si(R₉)₃, halogen, NO₂, alkaryl,sulfoxyl, sulfonyl, thio, substituted sulfoxyl, substituted sulfonyl, orsubstituted thio, wherein the substituents are selected from the groupconsisting of alkyl, aryl, or alkaryl;

each R₉ is, independently, alkyl having 1 to about 10 carbon atoms, oraryl having 6 to about 10 carbon atoms;

X₁ is O or S; comprising the steps of:

(a) providing a compound having the Formula II: ##STR10## wherein: eachR₁, is, independently, H, --OH, --F, or --O--X₃ --D;

X₃ is alkyl having from 1 to 10 carbons;

D is H, amino, protected amino, alkyl substituted amino, imidazole, or(--O--X₃)_(p), where p is 1 to about 10;

each X₂ is O or S;

R₃ and R_(3a) are each hydrogen, a hydroxyl protecting group, or alinker connected to a solid support;

each B, independently, is a naturally occurring or non-naturallyoccurring nucleobase or a protected naturally occurring or non-naturallyoccurring nucleobase;

n is 0 to about 50;

each Q is --X₁ H or --X₁ --CH₂ --CH═CH--CH₂ --Z;

R₅ is --N(R₆)₂, or a heterocycloalkyl or heterocycloalkenyl ringcontaining from 4 to 7 atoms, and having up to 3 heteroatoms selectedfrom the group consisting of nitrogen, sulfur, and oxygen;

R₆ is straight or branched chain alkyl having from 1 to 10 carbons;

(b) reacting the compound of Formula II with a compound having theFormula III: ##STR11## wherein R_(3a) is hydrogen;and R₂ is a hydroxylprotecting group, or a linker connected to a solid support, providedthat R₂ and R₃ are not both simultaneously a linker connected to a solidsupport;

to form the oligomeric compound.

The methods of the present invention are useful for the preparation ofoligomeric compounds containing monomeric subunits that are joined by avariety of linkages, including phosphite, phosphodiester,phosphorothioate, and/or phosphorodithioate linkages. As used herein,the term "oligomeric compound" is used to refer to compounds containinga plurality of monomer subunits that are joined by phosphite,phosphodiester, phosphorothioate, and/or phosphorodithioate linkages.Oligomeric compounds include oligonucleotides, their analogs, andsynthetic oligonucleotides. Monomer or higher order synthons havingFormulas II or III above include both native (i.e., naturally occurring)and synthetic (e.g., modified native of totally synthetic) nucleosides.

In preferred embodiments, compounds of Formula II and Formula III arereacted to produce compounds of Formula IV. Methods for couplingcompounds of Formula II and Formula III of the invention include bothsolution phase and solid phase chemistries. Representative solutionphase techniques are described in U.S. Pat. No. 5,210,264, which isassigned to the assignee of the present invention. In preferredembodiments, the methods of the present invention are employed for usein iterative solid phase oligonucleotide synthetic regimes.Representative solid phase techniques are those typically employed forDNA and RNA synthesis utilizing standard phosphoramidite chemistry,(see, e.g., Protocols For Oligonucleotides And Analogs, Agrawal, S.,ed., Humana Press, Totowa, N.J., 1993). A preferred synthetic solidphase synthesis utilizes phosphoramidites as activated phosphatecompounds. In this technique, a phosphoramidite monomer is reacted witha free hydroxyl on the growing oligomer chain to produce an intermediatephosphite compound, which is subsequently oxidized to the P^(V) stateusing standard methods. This technique is commonly used for thesynthesis of several types of linkages including phosphodiester,phosphorothioate, and phosphorodithioate linkages.

Typically, the first step in such a process is attachment of a firstmonomer or higher order subunit containing a protected 5'-hydroxyl to asolid support, usually through a linker, using standard methods andprocedures known in the art. The support-bound monomer or higher orderfirst synthon is then treated to remove the 5'-protecting group, to forma compound of Formula III wherein R₂ is a linker connected to a solidsupport. Typically, this is accomplished by treatment with acid. Thesolid support bound monomer is then reacted with a compound of FormulaII to form a compound of Formula IV, which has a phosphite orthiophosphite linkage of Formula IX. In preferred embodiments, synthonsof Formula II and Formula III are reacted under anhydrous conditions inthe presence of an activating agent such as, for example, 1H-tetrazole,5-(4-nitrophenyl)-1H-tetrazole, or diisopropylamino tetrazolide.

In preferred embodiments, phosphite or thiophosphite compoundscontaining a linkage of Formula IX are oxidized as shown below toproduce compounds having a linkage of Formula XI, where X₁ and X₂ caneach be O or S: ##STR12##

Choice of oxidizing agent will determine whether the linkage of FormulaIX is oxidized to a phosphotriester, thiophosphotriester, or adithiophosphotriester linkage.

It is generally preferable to perform a capping step, either prior to orafter oxidation of the phosphite triester, thiophosphite triester, ordithiophosphite triester. Such a capping step is generally known toprovide benefits in the prevention of shortened oligomer chains, byblocking chains that have not reacted in the coupling cycle. Onerepresentative reagent used for capping is acetic anhydride. Othersuitable capping reagents and methodologies can be found in U.S. Pat.No. 4,816,571, issued Mar. 28, 1989.

Treatment with an acid removes the 5'-hydroxyl protecting group, andthus transforms the solid support bound oligomer into a further compoundof Formula III wherein R_(3a) is hydrogen, which can then participate inthe next synthetic iteration; i.e., which can then be reacted with afurther compound of Formula II. This process is repeated until anoligomer of desired length is produced.

The completed oligomer is then cleaved from the solid support. Thecleavage step, which can precede or follow deprotection of protectedfunctional groups, will yield a compound having the Formula I wherein R₂is hydrogen. During cleavage, the linkages between monomeric subunitsare converted from phosphotriester, thiophosphotriester, ordithiophosphotriester linkages to phosphodiester, phosphorothioate, orphosphorodithioate linkages. This conversion is effected through theloss of an oxygen or sulfur protecting group of Formula Z--CH₂--CH═CH--CH₂ --. Depending upon the species Z, it is believed that theloss of an oxygen or sulfur protecting group occurs via either aδ-elimination mechanism, or a δ-fragmentation mechanism.

While not wishing to be bound by a particular theory, it is believedthat the loss of the oxygen or sulfur protecting group where Z is anon-silyl electron withdrawing group occurs via a δ-eliminationmechanism, illustrated in Scheme I below: ##STR13##

In this mechanism, a base first abstracts an acidic proton from thecarbon atom adjacent to electron withdrawing group Z. The resonantmovement of electrons as depicted in Scheme I above is believed to causethe loss of the oxygen or sulfur protecting group via a δ-elimination,thereby forming a phosphodiester, phosphorothioate, orphosphorodithioate linkage. The other product of the deprotection is a1-substituted-1,3-butadiene, having electron withdrawing substituent Zat the 1-position.

Substituent Z can be an electron withdrawing group selected such that itfacilitates the abstraction of a proton from the adjacent carbon atom byresonance, inductive, or other electron withdrawing mechanisms.Accordingly, Z can be any of a variety of electron withdrawingsubstituents, provided that it does not otherwise interfere with themethods of the invention. Preferred non-silyl electron withdrawing Zgroups include CN, halogens, NO₂, alkaryl groups, sulfoxyl groups,sulfonyl groups, thio groups, substituted sulfoxyl groups, substitutedsulfonyl groups, or substituted thio groups, wherein the substituentsare selected from the group consisting of alkyl, aryl, or alkaryl. Inmore preferred embodiments Z is cyano.

Z can also be a trisubstituted silyl moiety, wherein the substituentsare alkyl, aryl or both. While not wishing to be bound by a particulartheory, it is believed that the loss of the oxygen or sulfur protectinggroup, where Z is such a trisubstituted silyl moiety, occurs via aδ-fragmentation mechanism, illustrated in Scheme II below: ##STR14## Inthis scheme, a nucleophile attacks the silyl silicon atom, and theresonant movement of electrons as depicted in Scheme II above isbelieved to cause the loss of the oxygen or sulfur protecting group viaa δ-fragmentation mechanism, thereby forming a phosphodiester,phosphorothioate, or phosphorodithioate linkage. The other products ofthe deprotection are 1,3-butadiene, and Nu--Si(R₃)₃.

A wide variety of bases can be used to initiate the δ-elimination of theoxygen or sulfur protecting group. These include aqueous ammoniumhydroxide, aqueous methylamine, or DBU (1,8-diazabicyclo5.4.0!undec-7-ene).

A wide variety of nucleophiles can be used to initiate theδ-fragmentation of the oxygen or sulfur protecting group. These includeammonium hydroxide, fluoride ion, alkyl amines, aqueous bases, and alkylamines in combination with ammonium hydroxide. The resulting productsinclude phosphate, phosphorothioate, and phosphorodithioate containingcompounds.

Contact with fluoride ion preferably is effected in a solvent such astetrahydrofuran, acetonitrile, dimethoxyethane, or water. Fluoride ionpreferably is provided in the form of one or more salts selected fromtetraalkylammonium fluorides (e.g., tetrabutylammonium fluoride (TBAF)),potassium fluoride, or cesium fluoride.

Preferably, conditions for removal of the oxygen or sulfur protectinggroup via δ-elimination or δ-fragmentation also effect cleavage of theoligomeric compound from the solid support.

The methods of the present invention are applicable to the synthesis ofa wide variety of oligomeric compounds which contain phosphite,phosphodiester, phosphorothioate, or phosphorodithioate linkages. Asused herein, the term "oligomeric compound" denotes a polymeric compoundcontaining two or more monomeric subunits joined by such phosphite,phosphodiester, phosphorothioate, or phosphorodithioate linkages.

In preferred embodiments, the methods of the invention are used for thepreparation of oligonucleotides and their analogs. As used herein, theterm "oligonucleotide analog" means compounds that can contain bothnaturally occurring (i.e. "natural") and non-naturally occurring("synthetic") moieties, for example, nucleosidic subunits containingmodified sugar and/or nucleobase portions. Such oligonucleotide analogsare typically structurally distinguishable from, yet functionallyinterchangeable with, naturally occurring or synthetic wild typeoligonucleotides. Thus, oligonucleotide analogs include all suchstructures which function effectively to mimic the structure and/orfunction of a desired RNA or DNA strand, for example, by hybridizing toa target. The term synthetic nucleoside, for the purpose of the presentinvention, refers to a modified nucleoside. Representative modificationsinclude modification of a heterocyclic base portion of a nucleoside togive a non-naturally occurring nucleobase, a sugar portion of anucleoside, or both simultaneously.

Representative nucleobases include adenine, guanine, cytosine, uridine,and thymine, as well as other non-naturally occurring and naturalnucleobases such as xanthine, hypoxanthine, 2-aminoadenine, 6-methyl andother alkyl derivatives of adenine and guanine, 2-propyl and other alkylderivatives of adenine and guanine, 5-halo uracil and cytosine, 6-azouracil, cytosine and thymine, 5-uracil (pseudo uracil), 4-thiouracil,8-halo, oxa, amino, thiol, thioalkyl, hydroxyl and other 8-substitutedadenines and guanines, 5-trifluoromethyl and other 5-substituted uracilsand cytosines, 7-methylguanine. Further naturally and non naturallyoccurring nucleobases include those disclosed in U.S. Pat. No. 3,687,808(Merigan, et al.), in chapter 15 by Sanghvi, in Antisense Research andApplication, Ed. S. T. Crooke and B. Lebleu, CRC Press, 1993, inEnglisch et al., Angewandte Chemie, International Edition, 1991, 30,613-722 (see especially pages 622 and 623, and in the ConciseEncyclopedia of Polymer Science and Engineering, J. I. Kroschwitz Ed.,John Wiley & Sons, 1990, pages 858-859, Cook, P. D., Anti-Cancer DrugDesign, 1991, 6, 585-607. The term `nucleosidic base` is furtherintended to include heterocyclic compounds that can serve as likenucleosidic bases including certain `universal bases` that are notnucleosidic bases in the most classical sense but serve as nucleosidicbases. Especially mentioned as a universal base is 3-nitropyrrole.

Representative 2' sugar modifications (position R₁) amenable to thepresent invention include fluoro, O-alkyl, O-alkylamino, O-alkylalkoxy,protected O-alkylamino, O-alkylaminoalkyl, O-alkyl imidazole, andpolyethers of the formula (O-alkyl)_(m), where m is 1 to about 10.Preferred among these polyethers are linear and cyclic polyethyleneglycols (PEGs), and (PEG)-containing groups, such as crown ethers andthose which are disclosed by Ouchi, et al., Drug Design and Discovery1992, 9, 93, Ravasio, et al., J. Org. Chem. 1991, 56, 4329, and Delgardoet. al., Critical Reviews in Therapeutic Drug Carrier Systems 1992, 9,249. Further sugar modifications are disclosed in Cook, P. D., supra.Fluoro, O-alkyl, O-alkylamino, O-alkyl imidazole, O-alkylaminoalkyl, andalkyl amino substitution is described in U.S. patent application Ser.No. 08/398,901, filed Mar. 6, 1995, entitled Oligomeric Compounds havingPyrimidine Nucleotide(s) with 2' and 5' Substitutions.

Sugars having O-substitutions on the ribosyl ring are also amenable tothe present invention. Representative substitutions for ring O includeS, CH₂, CHF, and CF₂, see, e.g., Secrist, et al., Abstract 21, Program &Abstracts, Tenth International Roundtable, Nucleosides, Nucleotides andtheir Biological Applications, Park City, Utah, Sep. 16-20, 1992.

As used herein, the term "alkyl" includes but is not limited to straightchain, branch chain, and alicyclic hydrocarbon groups. Alkyl groups ofthe present invention may be substituted. Representative alkylsubstituents are disclosed in U.S. Pat. No. 5,212,295, at column 12,lines 41-50.

As used herein, the term "aralkyl" denotes alkyl groups which bear arylgroups, for example, benzyl groups. The term "alkaryl" denotes arylgroups which bear alkyl groups, for example, methylphenyl groups. "Aryl"groups are aromatic cyclic compounds including but not limited tophenyl, naphthyl, anthracyl, phenanthryl, pyrenyl, and xylyl.

As used herein, the term "heterocycloalkyl" denotes an alkyl ring systemhaving one or more heteroatoms (i.e., non-carbon atoms). Preferredheterocycloalkyl groups include, for example, morpholino groups. As usedherein, the term "heterocycloalkenyl" denotes a ring system having oneor more double bonds, and one or more heteroatoms. Preferredheterocycloalkenyl groups include, for example, pyrrolidino groups.

In some preferred embodiments of the invention R₂, R₃ or R_(3a) can be alinker connected to a solid support. Solid supports are substrates whichare capable of serving as the solid phase in solid phase syntheticmethodologies, such as those described in Caruthers U.S. Pat. Nos.4,415,732; 4,458,066; 4,500,707; 4,668,777; 4,973,679; and 5,132,418;and Koster U.S. Pat. No. 4,725,677 and U.S. Pat. No. Re. 34,069. Linkersare known in the art as short molecules which serve to connect a solidsupport to functional groups (e.g., hydroxyl groups) of initial synthonmolecules in solid phase synthetic techniques. Suitable linkers aredisclosed in, for example, Oligonucleotides And Analogues A PracticalApproach, Ekstein, F. Ed., IRL Press, N.Y., 1991, Chapter 1, pages 1-23.

Solid supports according to the invention include those generally knownin the art to be suitable for use in solid phase methodologies,including, for example, controlled pore glass (CPG), oxalyl-controlledpore glass (see, e.g., Alul, et al., Nucleic Acids Research 1991, 19,1527), TentaGel Support--an aminopolyethyleneglycol derivatized support(see, e.g., Wright, et al., Tetrahedron Letters 1993, 34, 3373) andPoros--a copolymer of polystyrene/divinylbenzene.

In some preferred embodiments of the invention R₂, R₃ or R_(3a) can be ahydroxyl protecting group. A wide variety of hydroxyl protecting groupscan be employed in the methods of the invention. Preferably, theprotecting group is stable under basic conditions but can be removedunder acidic conditions. In general, protecting groups render chemicalfunctionalities inert to specific reaction conditions, and can beappended to and removed from such functionalities in a molecule withoutsubstantially damaging the remainder of the molecule. Representativehydroxyl protecting groups are disclosed by Beaucage, et al.,Tetrahedron 1992, 48, 2223-2311, and also in Greene and Wuts, ProtectiveGroups in Organic Synthesis, Chapter 2, 2d ed, John Wiley & Sons, NewYork, 1991. Preferred protecting groups used for R₂, R₃ and R_(3a)include dimethoxytrityl (DMT), monomethoxytrityl, 9-phenylxanthen-9-yl(Pixyl) and 9-(p-methoxyphenyl)xanthen-9-yl (Mox). The R₂ or R₃ groupcan be removed from oligomeric compounds of the invention by techniqueswell known in the art to form the free hydroxyl. For example,dimethoxytrityl protecting groups can be removed by protic acids such asformic acid, dichloroacetic acid, trichloroacetic acid, p-toluenesulphonic acid or with Lewis acids such as for example zinc bromide. Seefor example, Greene and Wuts, supra.

In some preferred embodiments of the invention amino groups are appendedto alkyl or other groups, such as, for example, 2'-alkoxy groups (e.g.,where R₁ is alkoxy). Such amino groups are also commonly present innaturally occurring and non-naturally occurring nucleobases. It isgenerally preferred that these amino groups be in protected form duringthe synthesis of oligomeric compounds of the invention. Representativeamino protecting groups suitable for these purposes are discussed inGreene and Wuts, Protective Groups in Organic Synthesis, Chapter 7, 2ded, John Wiley & Sons, New York, 1991. Generally, as used herein, theterm "protected" when used in connection with a molecular moiety such as"nucleobase" indicates that the molecular moiety contains one or morefunctionalities protected by protecting groups.

Sulfurizing agents used during oxidation to form phosphorothioate andphosphorodithioate linkages include Beaucage reagent (see e.g. Iyer, R.P., et.al., J. Chem. Soc., 1990, 112, 1253-1254, and Iyer, R. P.,et.al., J. Org. Chem., 1990, 55, 4693-4699); tetraethylthiuram disulfide(see e.g., Vu, H., Hirschbein, B. L., Tetrahedron Lett., 1991, 32,3005-3008); dibenzoyl tetrasulfide (see e.g., Rao, M. V., et.al.,Tetrahedron Lett., 1992, 33, 4839-4842); di(phenylacetyl)disulfide (seee.g., Kamer, P. C. J., Tetrahedron Lett., 1989, 30, 6757-6760); sulfur,sulfur in combination with ligands like triaryl, trialkyl, triaralkyl,or trialkaryl phosphines.

Useful oxidizing agents used to form the phosphodiester orphosphorothioate linkages include iodine/tetrahydrofuran/water/pyridineor hydrogen peroxide/water or tert-butyl hydroperoxide or any peracidlike m-chloroperbenzoic acid. In the case of sulfurization the reactionis performed under anhydrous conditions with the exclusion of air, inparticular oxygen whereas in the case of oxidation the reaction can beperformed under aqueous conditions.

Oligonucleotides or oligonucleotide analogs according to the presentinvention hybridizable to a specific target preferably comprise fromabout 5 to about 50 monomer subunits. It is more preferred that suchcompounds comprise from about 10 to about 30 monomer subunits, with 15to 25 monomer subunits being particularly preferred. When used as"building blocks" in assembling larger oligomeric compounds (i.e., assynthons of Formula II), smaller oligomeric compounds are preferred.Libraries of dimeric, trimeric, or higher order compounds of generalFormula II can be prepared for use as synthons in the methods of theinvention. The use of small sequences synthesized via solution phasechemistries in automated synthesis of larger oligonucleotides enhancesthe coupling efficiency and the purity of the final oligonucleotides.See for example: Miura, K., et al., Chem. Pharm. Bull., 1987, 35,833-836; Kumar, G., and Poonian, M. S., J. Org. Chem., 1984, 49,4905-4912; Bannwarth, W., Helvetica Chimica Acta, 1985, 68, 1907-1913;Wolter, A., et al., nucleosides and nucleotides, 1986, 5, 65-77.

In one aspect of the invention, the compounds of the invention are usedto modulate RNA or DNA, which code for a protein whose formation oractivity it is desired to modulate. The targeting portion of thecomposition to be employed is, thus, selected to be complementary to thepreselected portion of DNA or RNA, that is to be hybridizable to thatportion.

In preferred embodiments of the methods of the invention, the compoundof Formula II is prepared by reaction of a protected nucleoside havingFormula V: ##STR15## and a phosphine compound of Formula VI:

    (R.sub.5).sub.2 P--X.sub.1 --CH.sub.2 --CH═CH--CH.sub.2 --ZVI

in the presence of an acid. Suitable acids include those known in theart to be useful for coupling of phosphoramidites, including, forexample, diisopropylammonium tetrazolide.

Compounds of Formula VI are preferably prepared by reacting an alcoholhaving the formula HOCH₂ CH═CHCH₂ Z with phosphorus trichloride, andreacting the resultant product, Cl₂ PX₁ CH₂ CH═CHCH₂ Z, with at leasttwo equivalents of an amine having the formula (R₆)₂ N!₂ NH. Each of theR₆ groups can be the same or different, and are preferably alkyl having1 to about 10 carbon atoms, more preferably 1 to 6 carbon atoms, with 3carbon atoms, and particularly isopropyl groups, being especiallypreferred.

X₁ and X₂ can each independently be O or S. Thus, compounds havingchiral phosphorus linkages are contemplated by the present invention.See Stec, W. J., and Lesnikowski, Z. J., in Methods in Molecular BiologyVol. 20: Protocols for Oligonucleotides and Analogs, S. Agrawal, Ed.,Humana Press, Totowa, N.J. (1993), at Chapter 14. See also Stec, W. J.et al., Nucleic Acids Research, Vol. 19, No. 21, 5883-5888 (1991); andEuropean Patent Application EP 0 506 242 A1.

Also provided in preferred embodiments of the invention are compoundshaving the general Formula VII:

    A--X.sub.1 --CH.sub.2 --CH═CH--CH.sub.2 --Z            VII

wherein X₁, A, and Z are as defined above.

The oligomeric compounds of the invention can be used in diagnostics,therapeutics and as research reagents and kits. They can be used inpharmaceutical compositions by including a suitable pharmaceuticallyacceptable diluent or carrier. They further can be used for treatingorganisms having a disease characterized by the undesired production ofa protein. The organism should be contacted with an oligonucleotidehaving a sequence that is capable of specifically hybridizing with astrand of nucleic acid coding for the undesirable protein. Treatments ofthis type can be practiced on a variety of organisms ranging fromunicellular prokaryotic and eukaryotic organisms to multicellulareukaryotic organisms. Any organism that utilizes DNA-RNA transcriptionor RNA-protein translation as a fundamental part of its hereditary,metabolic or cellular control is susceptible to therapeutic and/orprophylactic treatment in accordance with the invention. Seeminglydiverse organisms such as bacteria, yeast, protozoa, algae, all plantsand all higher animal forms, including warm-blooded animals, can betreated. Further, each cell of multicellular eukaryotes can be treated,as they include both DNA-RNA transcription and RNA-protein translationas integral parts of their cellular activity. Furthermore, many of theorganelles (e.g., mitochondria and chloroplasts) of eukaryotic cellsalso include transcription and translation mechanisms. Thus, singlecells, cellular populations or organelles can also be included withinthe definition of organisms that can be treated with therapeutic ordiagnostic oligonucleotides.

As will be recognized, the steps of the methods of the present inventionneed not be performed any particular number of times or in anyparticular sequence. Additional objects, advantages, and novel featuresof this invention will become apparent to those skilled in the art uponexamination of the following examples thereof, which are intended to beillustrative and not intended to be limiting.

EXAMPLE 1

1-Chloro-2-butene-4-ol

2-Butene-1,4-diol (600 g, 6.81 mol) was added to a 5-liter three-neckedround bottomed flask equipped with a condenser, a pressure equalizingaddition funnel and a mechanical stirrer. To this was added anhydrousether (1400 mL) and pyridine (604.9 mL, 7.49 mol). The reaction flaskwas cooled to 0° C. and thionyl chloride (545.6 mL, 7.49 mol) was addeddropwise over a period of 2.5 hours. After the addition was complete,the reaction mixture was allowed to warm to room temperature and stirredovernight. The reaction mixture was then poured into 500 mL of ice waterand extracted with ether (2×400 mL). The combined ether extracts werewashed with saturated sodium bicarbonate followed by brine and dried(Na₂ SO₄). Concentration of the dried extracts gave the title compoundwhich was used in the next step without further purification.

EXAMPLE 2

4-Cyano-2-butene-1-ol

1-Chloro-2-butene-4-ol (230 g, 2.14 mol) was dissolved in anhydrousacetonitrile (1250 mL) and added to a 3 L round bottomed flask under anatmosphere of argon. Potassium cyanide (825 g, 12.5 mol) was added allat once and the reaction was stirred at room temperature for 3 hours.NaI (16 g, 0.054 mol) was added and the reaction mixture was stirredovernight at room temperature. The reaction mixture was filtered and thesolid washed with ethyl acetate (800 mL). Concentration of the filtratein vacuo afforded an oil which was triturated with ether (750 mL). Thismixture was filtered and the clear filtrate concentrated. The crudeproduct was distilled using a short path to give the title compound, bp89°-91° C. at 0.2 mm Hg. IR (neat) cm⁻¹ : 3400, 2900, 2250, 1650.

EXAMPLE 3

4-Cyano-2-butenyl-N,N,N',N'-tetraisopropylphosphorodiamidite

A 500 mL three-necked flask equipped with a magnetic stirrer, a glassstopper and an inlet for argon is assembled under an atmosphere ofargon. All the glassware is dried in an oven at 120° C. for 1 hour.Anhydrous ether (150 mL) and phosphorous trichloride (67.5 mmol) isadded to the flask. 4-Cyanobutene-1-ol (50 mmol) in ether (50 mL) isadded to the reaction flask slowly with stirring at 0° C. (ice bath)using a pressure-equalized addition funnel. After the addition iscomplete, the ice bath is removed and the reaction is stirred for threehours at room temperature. The reaction mixture is then transferred to a500 mL flask and concentrated under reduced pressure.

To the product in anhydrous ether (200 mL) is added diisopropylamine(57.7 mL) at 0° C. under argon. After the addition is complete, stirringis continued at room temperature for 16 hours. The reaction mixture isfiltered and concentrated to afford the title compound.

EXAMPLE 4

Preparation of protected phosphoramidite monomers

A. 5'-O-DMT-thymidine-3'-O-(4-cyano-2-butenylN,N-diisopropylphosphoramidite)

A 250 mL two-necked flask equipped with a magnetic stirrer, a gas inletfor argon, and a septum is assembled under an atmosphere of argon. Allthe glassware is dried at 120° C. for 1 hour. 5'-O-DMT-thymidine (7mmol) and 5-(4-nitrophenyl)1H-tetrazole (5.6 mmol) and anhydrousacetonitrile (50 mL) is added to the flask. To this stirred mixture atroom temperature is added a solution of4-cyano-2-butenyl-N,N,N',N'-tetraisopropylphosphorodiamidite (10.5 mmol)in acetonitrile (50 mL). After stirring for two hours, the reactionmixture is filtered and the filtrate diluted with ethyl acetate (100mL), washed once with cold saturated sodium bicarbonate solution, brineand dried (MgSO₄). The dried solution is concentrated under reducedpressure and purified by silica gel flash column chromatography to givethe title compound.

B. N2-Isobutyryl-5'-O-DMT-2'-deoxyguanosine-3'-O-(4-cyano-2-butenylN,N-diisopropylphosphoramidite)

A 250 mL two-necked flask equipped with a magnetic stirrer, a gas inletfor argon, and a septum is assembled under an argon atmosphere. All theglassware is dried at 120° C. for 1 hour. The flask is charged withN2-isobutyryl-5'-O-DMT-2'-deoxyguanosine (5 mmol) and diisopropylammonium tetrazolide (4 mmol). Anhydrous acetonitrile (50 mL) is added.To this stirred mixture at room temperature is added a solution of4-cyano-2-butenyl-N,N,N',N'-tetraisopropylphosphorodiamidite (7.5 mmol)in acetonitrile (50 mL). After stirring for two hours, the reactionmixture is filtered and the filtrate diluted with ethyl acetate (100mL), washed once with cold saturated sodium bicarbonate solution, brineand dried (MgSO₄). The dried solution is concentrated under reducedpressure to afford the product which is purified by silica gel flashcolumn chromatography.

C. N6-Benzoyl-5'-O-DMT-2'-deoxyadenosine-3'-O-(4-cyano-2-butenylN,N-diisopropylphosphoramidite)

A 250 mL two-necked flask equipped with a magnetic stirrer, a gas inletfor argon, and a septum is assembled under an atmosphere of argon. Allthe glassware is dried at 120° C. for 1 hour. The flask is charged withN6-benzoyl-5'-O-DMT-2'-deoxyadenosine (5 mmol) and diisopropylammoniumtetrazolide (4 mmol). Anhydrous acetonitrile (50 mL) is added. To thisstirred mixture at room temperature is added a solution of4-cyano-2-butenyl-N,N,N',N'-tetraisopropylphosphorodiamidite (6 mmol) inacetonitrile (50 mL). After stirring for two hours, the reaction mixtureis filtered and concentrated to a residue which is purified by silicagel flash column chromatography to give the title compound.

D. N4-Benzoyl-5'-O-DMT-2'-deoxycytidine-3'-O-(4-cyano-2-butenylN,N-diisopropylphosphoramidite)

A 250 mL two-necked flask equipped with a magnetic stirrer, a gas inletfor argon, and a septum is assembled under an atmosphere of argon. Allthe glassware is dried at 120° C. for 1 hour. The flask is charged withN4-benzoyl-5'-O-DMT-2'-deoxycytidine (5 mmol) and diisopropylammoniumtetrazolide (4 mmol). Anhydrous acetonitrile (50 mL) is added. To thisstirred mixture at room temperature is added a solution of4-cyano-2-butenyl-N,N,N',N'-tetraisopropylphosphorodiamidite (7.5 mmol)in acetonitrile (50 mL). After stirring for two hours, the reactionmixture is filtered and the filtrate diluted with ethyl acetate (100mL), washed once with cold saturated sodium bicarbonate solution, brineand dried (MgSO₄). The dried solution is concentrated under reducedpressure to afford the product which is purified by silica gel flashcolumn chromatography.

EXAMPLE 5

Coupling Procedures

A. Synthesis of T-T phosphorothioate dimer

100 milligram (4 mmole) of 5'-O-DMT-thymidine bonded to CPG (controlledpore glass) through an ester linkage is transferred to a glass reactor,and a dichloromethane solution of 2% dichloroacetic acid (volume/volume)is added to deprotect the 5'-hydroxyl group. The product is washed withdichloromethane and then with acetonitrile. Then, a 0.2M solution of5'-O-DMT-thymidine-3'-O-(4-cyano-2-butenylN,N-diisopropylphosphoramidite) in acetonitrile and a 0.4M solution of1H-tetrazole in acetonitrile is added, and reacted at room temperaturefor 5 minutes. The product is washed with acetonitrile, and then a 0.05Msolution of Beaucage reagent in acetonitrile is added and reacted atroom temperature for 5 minutes. This sulfurization step is repeated onemore time for 5 minutes. The support is washed with acetonitrile andthen a solution of acetic anhydride/lutidine/THF (1:1:8), and N-methylimidazole/THF is added to cap the unreacted 5'-hydroxyl group. Theproduct is washed with acetonitrile.

The carrier containing the compound is treated with 30% aqueous ammoniumhydroxide solution for 24 hours at 65° C. The aqueous solution isfiltered, concentrated under reduced pressure to give thephosphorothioate dimer of T-T.

B. Synthesis of C-T phosphorothioate dimer

5'-O-DMT-thymidine (100 mg, 4 mmole) bonded to CPG (controlled poreglass) through an ester linkage is transferred to a glass reactor, and aCH₂ CHl₂ solution of 2% dichloroacetic acid (v/v) is added to deprotectthe 5'-hydroxyl group. The product is washed with acetonitrile. Then, a0.2M solution ofN4-Benzoyl-5'-O-DMT-2'-deoxycytidine-3'-O-(4-cyano-2-butenylN,N-diisopropylphosphoramidite) in acetonitrile and a 0.4M solution of1H-tetrazole in acetonitrile is added, and reacted at room temperaturefor 5 minutes. The product is washed with acetonitrile, and then a 0.05Msolution of Beaucage reagent in acetonitrile is added and reacted atroom temperature for 5 minutes. This sulfurization step is repeated onemore time for 5 minutes. The support is washed with acetonitrile andthen a solution of acetic anhydride/lutidine/THF (1:1:8), and N-methylimidazole/THF is added to cap the unreacted 5'-hydroxyl groups. Theproduct is washed with acetonitrile.

The carrier containing the compound is treated with 30% aqueous ammoniumhydroxide solution for 24 hours at 65° C. The aqueous solution isfiltered, concentrated under reduced pressure to give thephosphorothioate dimer of dC-T.

C. Synthesis of T-T phosphodiester dimer

5'-O-DMT-thymidine (100 mg, 4 mmole) bonded to CPG through an esterlinkage is transferred to a glass reactor, and a CH₂ CHl₂ solution of 2%dichloroacetic acid (v/v) is added to deprotect the 5'-hydroxyl group.The product is washed with CH₂ CHl₂ and then with acetonitrile. Then, a0.2M solution of 5'-O-DMT-thymidine-3'-O-(4-cyano-2-butenylN,N-diisopropylphosphoramidite) in acetonitrile and a 0.4M solution of1H-tetrazole in acetonitrile is added, and reacted at room temperaturefor 5 minutes. The product is washed with acetonitrile, and then 0.1Miodine in water/pyridine/THF (2:20:80, v/v/v) is added and reacted atroom temperature for 5 minutes. The support is washed with acetonitrileand then a solution of acetic anhydride/lutidine/THF (1:1:8, v/v/v), andN-methyl imidazole/THF is added to cap the unreacted 5'-hydroxyl groups.The product is washed with acetonitrile.

The carrier containing the compound is treated with 30% aqueous ammoniumhydroxide solution for 24 hours at 65° C. The aqueous solution isfiltered, concentrated under reduced pressure to give the T-Tphosphodiester dimer.

D. Synthesis of 5'-TTTTTTT-3'- phosphorothioate heptamer

5'-O-DMT-thymidine (50 mg, 2 mmole) bonded to CPG through an esterlinkage transferred to a glass reactor, and a CH₂ Cl₂ solution of 2%dichloroacetic acid (v/v) is added to deprotect the 5'-hydroxyl group.The product is washed with acetonitrile. Then, a 0.2M solution of5'-O-DMT-thymidine-3'-O-(4-cyano-2-butenylN,N-diisopropylphosphoramidite) in acetonitrile and a 0.4M solution of1H-tetrazole in acetonitrile is added, and reacted at room temperaturefor 5 minutes. The product is washed with acetonitrile, and then a 0.05Msolution of Beaucage reagent in acetonitrile is added and reacted atroom temperature for 5 minutes. This sulfurization step is repeated onemore time for 5 minutes. The support is washed with acetonitrile andthen a solution of acetic anhydride/lutidine/THF (1:1:8, v/v/v), andN-methyl imidazole/THF is added to cap the unreacted 5'-hydroxyl groups.The product is washed with acetonitrile.

This complete cycle is repeated five more times to get the completelyprotected thymidine heptamer. The carrier containing the compound istreated with 30% aqueous ammonium hydroxide solution for 90 minutes atroom temperature. The aqueous solution is filtered and concentratedunder reduced pressure to give the phosphorothioate heptamer of TTTTTTT.

E. Synthesis of 5'-d(GACT)-3'- phosphorothioate tetramer

5'-O-DMT-thymidine (50 mg, 2 mmole) bonded to CPG through an esterlinkage is taken in a glass reactor, and a CH₂ Cl₂ solution of 2%dichloroacetic acid (v/v) is added to deprotect the 5'-hydroxyl group.The product is washed with acetonitrile. Then, a 0.2M solution of5'-O-DMT-thymidine-3'-O-(4-cyano-2-butenylN,N-diisopropylphosphoramidite) in acetonitrile and a 0.4M solution of1H-tetrazole in acetonitrile is added, and reacted at room temperaturefor 5 minutes. The product is washed with acetonitrile, and then a 0.05Msolution of Beaucage reagent in acetonitrile is added and reacted atroom temperature for 5 minutes. This sulfurization step is repeated onemore time for 5 minutes. The support is washed with acetonitrile andthen a solution of acetic anhydride/lutidine/THF (1:1:8, v/v/v), andN-methyl imidazole/THF is added to cap the unreacted 5'-hydroxyl groups.The product is washed with acetonitrile.

A CH₂ Cl₂ solution of 2% dichloroacetic acid (volume/volume) is added todeprotect the 5'-hydroxyl groups. The product is washed withacetonitrile. Then, a 0.2M solution ofN4-benzoyl-5'-O-DMT-2'-deoxycytidine-3'-O-(4-cyano-2-butenylN,N-diisopropylphosphoramidite) in acetonitrile and a 0.4M solution of1H-tetrazole in acetonitrile is added, and reacted at room temperaturefor 5 minutes. The product is washed with acetonitrile, and then a 0.05Msolution of Beaucage reagent in acetonitrile is added and reacted atroom temperature for 5 minutes. This sulfurization step is repeated onemore time for 5 minutes. The support is washed with acetonitrile andthen a solution of acetic anhydride/lutidine/THF (1:1:8, V/V/V), andN-methyl imidazole/THF is added to cap the unreacted 5'-hydroxyl groups.The product is washed with acetonitrile.

A CH₂ Cl₂ solution of 2% dichloroacetic acid (v/v) is added to deprotectthe 5'-hydroxyl group. The product is washed with acetonitrile. Then, a0.2M solution ofN6-benzoyl-5'-O-DMT-2'-deoxyadenosine-3'-O-(4-cyano-2-butenylN,N-diisopropylphosphoramidite) in anhydrous acetonitrile and a 0.4Msolution of 1H-tetrazole in acetonitrile is added, and reacted at roomtemperature for 5 minutes. The product is washed with acetonitrile, andthen a 0.05M solution of Beaucage reagent in acetonitrile is added andreacted at room temperature for 5 minutes. This sulfurization step isrepeated one more time for 5 minutes. The support is washed withacetonitrile and then a solution of acetic anhydride/lutidine/THF(1:1:8, v/v/v), and N-methyl imidazole/THF is added to cap the unreacted5'-hydroxyl groups. The product is washed with acetonitrile.

A dichloromethane solution of 2% dichloroacetic acid (volume/volume) isadded to deprotect the 5'-hydroxyl group. The product is washed withacetonitrile. Then, a 0.2M solution ofN2-isobutyryl-5'-O-DMT-2'-deoxyguanosine-3'-O-(4-cyano-2-butenylN,N-diisopropylphosphoramidite) in acetonitrile and a 0.4M solution of1H-tetrazole in acetonitrile is added, and reacted at room temperaturefor 5 minutes. The product is washed with acetonitrile, and then a 0.05Msolution of Beaucage reagent in acetonitrile is added and reacted atroom temperature for 5 minutes. This sulfurization step is repeated onemore time for 5 minutes. The support is washed with acetonitrile andthen a solution of acetic anhydride/lutidine/THF (1:1:8), and N-methylimidazole/THF is added to cap the unreacted 5'-hydroxyl group. Theproduct is washed with acetonitrile.

The carrier containing the compound is treated with 30% aqueous ammoniumhydroxide solution for 90 minutes at room temperature and then incubatedat 55° C. for 24 hour. The aqueous solution is filtered, concentratedunder reduced pressure to give a phosphorothioate tetramer of5'-dG-dA-dC-T-3'.

EXAMPLE 6

1-Trimethylsilylthioxy-4-cyano-2-butene

To a stirred mixture of 1-cyano-2,3-butadiene (0.1 mol) andtrimethylsilylthiol (0.1 mol) in anhydrous ether (300 ml) under argon,is added a catalytic amount of rhodium acetate at room temperature.After 24 hours the reaction mixture is filtered and concentrated to givethe title compound.

EXAMPLE 7

4-Cyano-2-butenyl-N,N,N',N'-tetraisopropylthiophosphorodiamidite

A 500 mL three-necked flask equipped with a magnetic stirrer, a glassstopper and an inlet for argon is assembled, after drying in an oven at120° C. for 1 hour. The flask is purged with argon while cooling and anatmosphere of argon is maintained inside the flask. Anhydrous ether (150mL) and phosphorous trichloride (67.5 mmol) is added to the flask.1-Trimethylsilyl-4-cyano-2-butene (50 mmol) in ether (50 mL) is slowlyadded to the reaction flask with stirring at 0° C. (ice bath) using apressure-equalized addition funnel. After the addition is complete, theice bath is removed and the reaction is stirred for three hours at roomtemperature. The reaction mixture is then transferred to a 500 mL flaskand concentrated under reduced pressure.

To the resulting residue in anhydrous ether (200 mL) is addeddiisopropylamine (57.7 mL) at 0° C. under argon. After the addition iscomplete, stirring is continued at room temperature for 16 hours. Thereaction mixture is filtered and concentrated and the resulting residuepurified by silica gel column chromatography to afford the titlecompound.

EXAMPLE 8

Preparation of protected phosphorothioamidite monomers

A. 5'-O-DMT-thymidine-3'-O-(4-cyano-2-butenylN,N-diisopropylthiophosphoramidite)

A 250 mL two-necked flask equipped with a magnetic stirrer, a glassstopper and an inlet for argon is assembled, after drying in an oven at120° C. for 1 hour. The flask is purged with argon while cooling and anatmosphere of argon is maintained inside the flask. 5'-O-DMT-thymidine(7 mmol) and 5-(4-nitrophenyl)1H-tetrazole (5.6 mmol) and anhydrousacetonitrile (50 mL) is added to the flask. To this stirred mixture atroom temperature is added a solution of4-cyano-2-butenyl-N,N,N',N'-tetraisopropylthiophosphorodiamidite (10.5mmol) in acetonitrile (50 mL). After stirring for two hours, thereaction mixture is filtered and the filtrate diluted with ethyl acetate(100 mL), washed once with cold aqueous solution of saturated sodiumbicarbonate, brine and dried (MgSO₄). The dried solution is filtered andthe filtrate is concentrated under reduced pressure and purified bysilica gel flash column chromatography to give the title compound.

B. N2-Isobutyryl-5'-O-DMT-2'-deoxyguanosine-3'-O-(4-cyano-2-butenylN,N-diisopropylthiophosphoramidite)

A 250 mL two-necked flask equipped with a magnetic stirrer, a glassstopper and an inlet for argon is assembled, after drying in an oven at120° C. for 1 hour. The flask is purged with argon while cooling and anatmosphere of argon is maintained inside the flask. The flask is chargedwith N2-isobutyryl-5'-O-DMT-2'-deoxyguanosine (5 mmol) and diisopropylammonium tetrazolide (4 mmol). Anhydrous acetonitrile (50 mL) is added.To this stirred mixture at room temperature is added a solution of4-cyano-2-butenyl-N,N,N',N'-tetraisopropylthiophosphorodiamidite (7.5mmol) in acetonitrile (50 mL). After stirring for two hours, thereaction mixture is filtered and the filtrate diluted with ethyl acetate(100 mL), washed once with cold saturated sodium bicarbonate solution,brine and dried (MgSO₄). The dried solution is filtered and theresulting filtrate is concentrated under reduced pressure. The resultingresidue is purified by silica gel flash column chromatography to givethe title compound.

C.N6-Benzoyl-5'-O-DMT-2'-deoxyadenosine-3'-O-(4-cyano-2-butenyl-N,N-diisopropylthiophosphoramidite)

A 250 mL two-necked flask equipped with a magnetic stirrer, a glassstopper and an inlet for argon is assembled, after drying in an oven at120° C. for 1 hour. The flask is purged with argon while cooling and anatmosphere of argon is maintained inside the flask. The flask is chargedwith N6-benzoyl-5'-O-DMT-2'-deoxyadenosine (5 mmol) anddiisopropylammonium tetrazolide (4 mmol). Anhydrous acetonitrile (50 mL)is added. To this stirred mixture at room temperature is added asolution of4-cyano-2-butenyl-N,N,N',N'-tetraisopropylthiophosphorodiamidite (6mmol) in acetonitrile (50 mL). After stirring for two hours, thereaction mixture is filtered and concentrated to a residue which ispurified by silica gel flash column chromatography to give the titlecompound.

D. N4-Benzoyl-5'-O-DMT-2'-deoxycytidine-3'-O-(4-cyano-2-butenylN,N-diisopropylthiophosphoramidite)

A 250 mL two-necked flask equipped with a magnetic stirrer, a glassstopper and an inlet for argon is assembled, after drying in an oven at120° C. for 1 hour. The flask is purged with argon while cooling and anatmosphere of argon is maintained inside the flask. The flask is chargedwith N4-benzoyl-5'-O-DMT-2'-deoxycytidine (5 mmol) anddiisopropylammonium tetrazolide (4 mmol). Anhydrous acetonitrile (50 mL)is added. To this stirred mixture at room temperature is added asolution of4-cyano-2-butenyl-N,N,N',N'-tetraisopropylthiophosphorodiamidite (7.5mmol) in acetonitrile (50 mL). After stirring for two hours, thereaction mixture is filtered and the filtrate diluted with ethyl acetate(100 mL), washed once with cold saturated sodium bicarbonate solutionand then brine. The organic layer is dried (MgSO₄), filtered, and thefiltrate concentrated under reduced pressure. The resulting residue ispurified by silica gel flash column chromatography to give the titlecompound.

EXAMPLE 9

Coupling Procedures

A. Synthesis of T-T phosphorodithioate dimer

100 milligram (4 mmole) of 5'-O-DMT-thymidine bonded to CPG (controlledpore glass) through an ester linkage (commercially available) istransferred to a glass reactor, and a dichloromethane solution of 2%dichloroacetic acid (volume/volume) is added to deprotect the5'-hydroxyl group. The product is washed with dichloromethane and thenwith acetonitrile. A 0.2M solution of5'-O-DMT-thymidine-3'-O-(4-cyano-2-butenylN,N-diisopropylthiophosphoramidite) in acetonitrile and a 0.4M solutionof 1H-tetrazole in acetonitrile is added, and reacted at roomtemperature for 5 minutes. The product is washed with acetonitrile, andthen a 0.05M solution of Beaucage reagent in acetonitrile is added andreacted at room temperature for 5 minutes. This sulfurization step isrepeated one more time for 5 minutes. The support is washed withacetonitrile and then a solution of acetic anhydride/lutidine/THF(1:1:8), and N-methyl imidazole/THF is added to cap any unreacted5'-hydroxyl groups. The product is washed with acetonitrile.

The CPG containing the compound is treated with 30% aqueous ammoniumhydroxide solution for 24 hours at 65° C. The aqueous solution isfiltered, concentrated under reduced pressure to give thephosphorodithioate T-T dimer.

B. Synthesis of C-T phosphorodithioate dimer

5'-O-DMT-thymidine (100 mg, 4 mmole) bonded to CPG through an esterlinkage is transferred to a glass reactor, and a CH₂ Cl₂ solution of 2%dichloroacetic acid (v/v) is added to deprotect the 5'-hydroxyl groups.The product is washed with acetonitrile. Then, a 0.2M solution ofN4-benzoyl-5'-O-DMT-2'-deoxycytidine-3'-O-(4-cyano-2-butenylN,N-diisopropylthiophosphoramidite) in acetonitrile and a 0.4M solutionof 1H-tetrazole in acetonitrile is added, and reacted at roomtemperature for 5 minutes. The product is washed with acetonitrile andthen a 0.05M solution of Beaucage reagent in acetonitrile is added andreacted at room temperature for 5 minutes. This sulfurization step isrepeated one more time for 5 minutes. The support is washed withacetonitrile and then a solution of acetic anhydride/lutidine/THF(1:1:8), and N-methyl imidazole/THF is added to cap the unreacted5'-hydroxyl groups. The product is washed with acetonitrile.

The carrier containing the compound is treated with 30% aqueous ammoniumhydroxide solution for 24 hours at 65° C. The aqueous solution isfiltered, concentrated under reduced pressure to give thephosphorodithioate dimer of dC-T.

C. Synthesis of T-T phosphorothioate dimer

5'-O-DMT-thymidine (100 mg, 4 mmole) bonded to CPG through an esterlinkage is transferred to a glass reactor, and a CH₂ Cl₂ solution of 2%dichloroacetic acid (v/v) is added to deprotect the 5'-hydroxyl group.The product is washed with CH₂ Cl₂ and then with acetonitrile. A 0.2Msolution of 5'-O-DMT-thymidine-3'-O-(4-cyano-2-butenylN,N-diisopropylthiophosphoramidite) in acetonitrile and a 0.4M solutionof 1H-tetrazole in acetonitrile is added, and reacted at roomtemperature for 5 minutes. The product is washed with acetonitrile, andthen 0.1M iodine in water/pyridine/THF (2:20:80, v/v/v) is added andreacted at room temperature for 5 minutes. The support is washed withacetonitrile and then a solution of acetic anhydride/lutidine/THF(1:1:8, v/v/v), and N-methyl imidazole/THF is added to cap the unreacted5'-hydroxyl groups. The product is washed with acetonitrile.

The CPG containing the compound is treated with 30% aqueous ammoniumhydroxide solution for 24 hours at 65° C. The aqueous solution isfiltered, concentrated under reduced pressure to give the T-Tphosphorothioate dimer.

D. Synthesis of 5'-TTTTTTT-3' phosphorodithioate heptamer

5'-O-DMT-thymidine (50 mg, 2 mmole) bonded to CPG through an esterlinkage is transferred to a glass reactor, and a CH₂ Cl₂ solution of 2%dichloroacetic acid (v/v) is added to deprotect the 5'-hydroxyl groups.The product is washed with acetonitrile. Then, a 0.2M solution of5'-O-DMT-thymidine-3'-O-(4-cyano-2-butenylN,N-diisopropylthiophosphoramidite) in acetonitrile and a 0.4M solutionof 1H-tetrazole in acetonitrile is added, and reacted at roomtemperature for 5 minutes. The product is washed with acetonitrile, andthen a 0.05M solution of Beaucage reagent in acetonitrile is added andreacted at room temperature for 5 minutes. This sulfurization step isrepeated one more time for 5 minutes. The support is washed withacetonitrile and then a solution of acetic anhydride/lutidine/THF(1:1:8, v/v/v), and N-methyl imidazole/THF is added to cap the unreacted5'-hydroxyl groups. The product is washed with acetonitrile.

This complete cycle is repeated five more times to get the completelyprotected thymidine heptamer. The carrier containing the compound istreated with 30% aqueous ammonium hydroxide solution for 90 minutes atroom temperature. The aqueous solution is filtered and concentratedunder reduced pressure to give the phosphorothioate heptamer of TTTTTTT.

E. Synthesis of 5'-d(GACT)-3' phosphorodithioate tetramer

5'-O-DMT-thymidine (50 mg, 2 mmole) bonded to CPG through an esterlinkage is taken in a glass reactor, and a CH₂ Cl₂ solution of 2%dichloroacetic acid (v/v) is added to deprotect the 5'-hydroxyl group.The product is washed with acetonitrile. Then, a 0.2M solution of5'-O-DMT-thymidine-3'-O-(4-cyano-2-butenylN,N-diisopropylthiophosphoramidite) in acetonitrile and a 0.4M solutionof 1H-tetrazole in acetonitrile is added, and reacted at roomtemperature for 5 minutes. The product is washed with acetonitrile, andthen a 0.05M solution of Beaucage reagent in acetonitrile is added andreacted at room temperature for 5 minutes. This sulfurization step isrepeated one more time for 5 minutes. The support is washed withacetonitrile and then a solution of acetic anhydride/lutidine/THF(1:1:8, v/v/v), and N-methyl imidazole/THF is added to cap the unreacted5'-hydroxyl groups. The product is washed with acetonitrile.

A CH₂ Cl₂ solution of 2% dichloroacetic acid (volume/volume) is added todeprotect the 5'-hydroxyl groups. The product is washed withacetonitrile. Then, a 0.2M solution ofN4-benzoyl-5'-O-DMT-2'-deoxycytidine-3'-O-(4-cyano-2-butenylN,N-diisopropylthiophosphoramidite) in acetonitrile and a 0.4M solutionof 1H-tetrazole in acetonitrile is added, and reacted at roomtemperature for 5 minutes. The product is washed with acetonitrile, andthen a 0.05M solution of Beaucage reagent in acetonitrile is added andreacted at room temperature for 5 minutes. This sulfurization step isrepeated one more time for 5 minutes. The support is washed withacetonitrile and then a solution of acetic anhydride/lutidine/THF(1:1:8, V/V/V), and N-methyl imidazole/THF is added to cap the unreacted5'-hydroxyl groups. The product is washed with acetonitrile.

A CH₂ Cl₂ solution of 2% dichloroacetic acid (v/v) is added to deprotectthe 5'-hydroxyl group. The product is washed with acetonitrile. Then, a0.2M solution ofN6-benzoyl-5'-O-DMT-2'-deoxyadenosine-3'-O-(4-cyano-2-butenylN,N-diisopropylthiophosphoramidite) in anhydrous acetonitrile and a 0.4Msolution of 1H-tetrazole in acetonitrile is added, and reacted at roomtemperature for 5 minutes. The product is washed with acetonitrile, andthen a 0.05M solution of Beaucage reagent in acetonitrile is added andreacted at room temperature for 5 minutes. This sulfurization step isrepeated one more time for 5 minutes. The support is washed withacetonitrile and then a solution of acetic anhydride/lutidine/THF(1:1:8, v/v/v), and N-methyl imidazole/THF is added to cap the unreacted5'-hydroxyl groups. The product is washed with acetonitrile.

A dichloromethane solution of 2% dichloroacetic acid (volume/volume) isadded to deprotect the 5'-hydroxyl group. The product is washed withacetonitrile. Then, a 0.2M solution ofN2-isobutyryl-5'-O-DMT-2'-deoxyguanosine-3'-O-(4-cyano-2-butenylN,N-diisopropylthiophosphoramidite) in acetonitrile and a 0.4M solutionof 1H-tetrazole in acetonitrile is added, and reacted at roomtemperature for 5 minutes. The product is washed with acetonitrile, andthen a 0.05M solution of Beaucage reagent in acetonitrile is added andreacted at room temperature for 5 minutes. This sulfurization step isrepeated one more time for 5 minutes. The support is washed withacetonitrile and then a solution of acetic anhydride/lutidine/THF(1:1:8), and N-methyl imidazole/THF is added to cap the unreacted5'-hydroxyl group. The product is washed with acetonitrile.

The carrier containing the compound is treated with 30% aqueous ammoniumhydroxide solution for 24 hours at 65° C. The aqueous solution isfiltered, concentrated under reduced pressure to give a phosphorothioatetetramer of 5'-dG-dA-dC-T-3'.

EXAMPLE 10

Standard Deprotection Procedures

A. Deprotection using NH₄ OH

A homothymidine phosphorothioate dodecamer was synthesized as per theprocedures of Example 5(D). The dodecamer was treated with saturatedNH₄₀ H for 24 hours at 65° C. to afford the completely deprotectedproduct as determined by mass spectroscopy.

B. Deprotection using CH₃ NH₂

A homothymidine phosphorothioate dodecamer was synthesized as per theprocedures of Example 5(D). The dodecamer was treated with CH₃ NH₂ at55° C. to afford the completely deprotected product as determined bymass spectroscopy.

EXAMPLE 11

Synthesis of a Phosphorothioate Homo T 20 mer

5'-O-DMT-thymidine bonded to CPG (controlled pore glass) through anester linkage (commercially available) is transferred to a glassreactor. The CPG bound 5'-O-DMT-thymidine was washed with acetonitrilefor 30 seconds followed by dichloromethane for 30 seconds. The CPG bound5'-O-DMT-thymidine was treated with dichloroacetic acid (3%) indichloromethane for 2 minutes followed by washing with acetonitrile for3 minutes.

The resulting detritylated thymidine bonded to CPG was reactedsimultaneously with equal volumes of5'-O-DMT-thymidine-3'-O-(4-cyano-2-butenylN,N-diisopropylphosphoramidite) (0.2M) in acetonitrile and 1H-tetrazole(0.4M) in acetonitrile at room temperature for 5 minutes. The reagentsare drained away and this step was repeated for an additional 5 minutes.The resulting T-T dimer bonded to CPG was washed with acetonitrile for30 seconds and oxidized with Beaucage reagent (0.5M) in acetonitrile for3 minutes. This sulfurization step was repeated for an additional 3minutes. The CPG was washed with acetonitrile for 30 seconds followed bytreatment with equal volumes of acetic anhydride/lutidine/THF (1:1:8),and N-methyl imidazole/THF for 1 minute to cap any unreacted sites. Theabove process of washing, detritylating, reacting with a monomersubunit, oxidizing and capping was repeated 18 times to synthesize the20 mer homo thymidine phosphorothioate oligomeric compound.

The CPG bound 20 mer was treated with 30% aqueous ammonium hydroxidesolution for 2 hours at room temperature. The aqueous solution wasfiltered, concentrated under reduced pressure to give thephosphorothioate homo T 20 mer.

The synthesis was run on a 1 μmole scale and the overall couplingefficiency was found to be greater than 99% as determined byspectrophotometric quantitation of releasedp,p'-dimethoxytriphenylmethyl cation.

EXAMPLE 12

Synthesis of a Phosphorothioate Mixed Sequence 20 mer(GCC-CAA-GCT-GGC-ATC-CGT-CA)

Following the procedures of Example 11, the mixed sequence 20 mer(GCC-CAA-GCT-GGC-ATC-CGT-CA) was synthesized using the protected monomersubunits of Example 4 (a,b,c, and d),5'-O-DMT-thymidine-3'-O-(4-cyano-2-butenylN,N-diisopropylphosphoramidite),N2-isobutyryl-5'-O-DMT-2'-deoxyguanosine-3'-O-(4-cyano-2-butenylN,N-diisopropylphosphoramidite),N6-benzoyl-5'-O-DMT-2'-deoxyadenosine-3'-O-(4-cyano-2-butenylN,N-diisopropylphosphoramidite),N4-Benzoyl-5'-O-DMT-2'-deoxycytidine-3'-O-(4-cyano-2-butenylN,N-diisopropylphosphoramidite).

The synthesis was carried out on a 1 μmole scale and the 20 mer wasdeprotected with aqueous NH₄ OH at room temperature for 1 hour followedby heating to 60° C. for 20 hours. The crude oligomer was purified byreverse-phase HPLC. The product was further characterized by capillarygel electrophoresis.

EXAMPLE 13

Synthesis of a Silyl-containing Nucleosides

The synthesis of representative silyl-containing nucleosides of theinvention is depicted below in Scheme 3: ##STR16##

Commercially available 1-acetoxy-1,3-butadiene (compound 1) is firstreacted with (R₉)₃ Si--H in the presence of Rh₂ Cl₂ (CO)₄ to producecompound 2. The hydroxyl group is deprotected using K₂ CO₃ in methanolto produce compound 3. Compound 3 is then reacted with PCl₃ in ether at0° C. to produce compound 4, which is further reacted withisopropylamine in ether to produce compound 5. Compound 5 is thenreacted with a 5'-DMT nucleoside 6 in the presence of tetrazole in CH₂Cl₂ at room temperature to yield synthon 7.

EXAMPLE 14

Preparation of 1,1,1-triphenyl-4-acetoxy-1-sila-2-butene

A solution of 1-acetoxy-1,3-butadiene (0.1 mol), triphenylsilane (0.1mol), and Rh₂ Cl₂ (CO)₄ (194.5 mg, 0.625 mol) in 100 mL of toluene wasstirred at room temperature under argon for 3 days. The reaction mixtureis treated with decolorizing charcoal, and the mixture boiled briefly.After cooling, the reaction mixture is filtered through Celite.Concentration of the solution will afford the title compound.

EXAMPLE 15

Preparation of 1,1,1-triphenyl-1-sila-2-butene-4-ol

The crude acetoxy compound from Example 14 is dissolved in 250 mLmethanol, and 25.0 g of potassium carbonate is added all at once. Afterstirring for 2 hours, the reaction mixture is filtered and concentrated.The concentrated residue is partitioned between 200/200 mL water/ethylacetate. The organic layer is removed, washed with brine, dried andconcentrated. The crude material is purified by flash chromatographyusing silica gel to afford the pure product.

EXAMPLE 16

Preparation of1,1,1-triphenyl-1-sila-2-butenyl-N,N-diisopropylbisphosphoramidite

A 500 mL three-necked flask equipped with a magnetic stirrer, a glassstopper and an inlet for argon is assembled under argon atmosphere. Allglassware are dried in an oven at 120° C. for 1 hour. The reaction flaskis charged with anhydrous ether (150 mL) and phosphorous trichloride(67.5 mmol). 1,1,1-triphenyl-1-sila-2-butene-4-ol (50 mmol) in ether (50mL) is added to the reaction flask slowly with stirring at 0° C. (icecooling) using pressure-equalized addition funnel. After addition iscomplete, ice bath is removed and the reaction is stirred for threehours. The reaction mixture then is transferred to a 500 mL flask andconcentrated under reduced pressure.

To this product in anhydrous ether (200 mL) is added diisopropylamine(57.7 mL) at 0° C. under argon. After the addition is complete, stirringis continued at room temperature for 16 hours (overnight). The reactionmixture is filtered and concentrated to afford the title compound.

EXAMPLE 17

Preparation of Protected Phosphoramidite Monomers

A.5'-O-(4,4'-dimethoxytrityl)thymidine-3'-O-(1,1,1-triphenyl-1-sila-2-butenyl-N,N-diisopropylphosphoramidite).

A 250 mL two-necked flask equipped with a magnetic stirrer, a gas inletfor argon, and a septum is assembled under an argon atmosphere. Allglassware are dried at 120° C. for 1 hour. The flask is charged with5'-O-(4,4'-dimethoxytrityl)thymidine (7 mmol) and5-(4-nitrophenyl)1H-tetrazole (5.6 mmol). Anhydrous acetonitrile (50 mL)is added. To this stirred mixture under argon at room temperature isadded a solution of 1,1,1-triphenyl-1-sila-2-butenylN,N-diisopropylphosphoramidite (10.5 mmol) in acetonitrile (50 mL).After stirring for two hours, the reaction mixture is filtered and thefiltrate diluted with ethyl acetate (100 mL), washed once with coldsaturated sodium bicarbonate solution, brine and dried (MgSO₄). Thedried solution is concentrated under reduced pressure to afford aviscous foamy liquid. The crude product is purified by flashchromatography using silica gel to afford the product. Triethylamine(1%) is used throughout the purification.

B. N² -Isobutyryl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxyguanosine-3'-O-(1,1,1-triphenyl-1-sila-2-butenyl-N,N-diisopropylphosphoramidite).

A 250 mL two-necked flask equipped with a magnetic stirrer, a gas inletfor argon, and a septum is assembled under an argon atmosphere. Allglassware are dried at 120° C. for 1 hour. The flask is charged withN2-Isobutyryl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxyguanosine (5 mmol) anddiisopropyl ammonium tetrazolide (4 mmol). Anhydrous acetonitrile (50mL) is added. To this stirred mixture under argon at room temperature isadded a solution of1,1,1-triphenyl-1-sila-2-butenyl-N,N-diisopropylphosphoramidite (7.5mmol) in acetonitrile (50 mL). After stirring for two hours, thereaction mixture is filtered and the filtrate diluted with ethyl acetate(100 mL), washed once with cold saturated sodium bicarbonate solution,brine and dried (MgSO₄). The dried solution is concentrated underreduced pressure to afford the product which is purified by flashchromatography using silica gel. Triethylamine (1%) is used throughoutthe purification.

C. N⁶-Benzoyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxyadenosine-3'-O-(1,1,1-triphenyl-1-sila-2-butenyl-N,N-diisopropylphosphoramidite).

A 250 mL two-necked flask equipped with a magnetic stirrer, a gas inletfor argon, and a septum is assembled under an argon atmosphere. Allglassware are dried at 120° C. for 1 hour. The flask is charged with N⁶-benzoyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxyadenosine (5 mmol) anddiisopropylammonium tetrazolide (4 mmol). Anhydrous acetonitrile (50 mL)is added. To this stirred mixture under argon at room temperature isadded a solution of1,1,1-triphenyl-1-sila-2-butenyl-N,N-diisopropylphosphoramidite (6 mmol)in acetonitrile (50 mL). After stirring for two hours, the reactionmixture is filtered and concentrated to afford the product which ispurified by flash chromatography using silica gel. Triethylamine (1%) isused throughout the purification.

D. N⁴-Benzoyl-5'-O-(4,4,-dimethoxytrityl)-2'-deoxycytidine-3'-O-(1,1,1-triphenyl-1-sila-2-butenyl-N,N-diisopropylphosphoramidite)

A 250 mL two-necked flask equipped with a magnetic stirrer, a gas inletfor argon, and a septum is assembled under an argon atmosphere. Allglassware are dried at 120° C. for 1 hour. The flask is charged withN4-benzoyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxycytidine (5 mmol) anddiisopropylammonium tetrazolide (4 mmol). Anhydrous acetonitrile (50 mL)is added. To this stirred mixture under argon at room temperature isadded a solution of 1,1,1-triphenyl-1-sila-2-butenylN,N-diisopropylphosphoramidite (7.5 mmol) in acetonitrile (50 mL). Afterstirring for two hours, the reaction mixture is filtered and thefiltrate diluted with ethyl acetate (100 mL), washed once with coldsaturated sodium bicarbonate solution, brine and dried (MgSO₄). Thedried solution is concentrated under reduced pressure to afford theproduct which is purified by flash chromatography using silica gel.Triethylamine (1%) is used throughout the purification.

EXAMPLE 18

Coupling Procedures

A. Synthesis of T-T phosphorothioate dimer

100 milligram (4 mmole) of 5'-O-dimethoxytritylthymidine bonded to CPG(controlled pore glass) through an ester linkage is taken in a glassreactor, and a dichloromethane solution of 2% dichloroacetic acid(volume/volume) is added to deprotect the 5'-hydroxyl group. The productis washed with dichloromethane and then with acetonitrile. Then, a 0.2Msolution of5'-O-(4,4'-dimethoxytrityl)thymidine-3'-O-(1,1,1-triphenyl-1-sila-2-butenyl-N,N-diisopropylphosphoramidite)in acetonitrile and a 0.4M solution of 1H-tetrazole in acetonitrile isadded, and reacted at room temperature for 5 minutes. The product iswashed with acetonitrile, and then a 0.05M solution of Beaucage reagentin acetonitrile is added and reacted at room temperature for 5 minutes.This sulfurization step is repeated one more time for 5 minutes. Thesupport is washed with acetonitrile and then a solution of aceticanhydride/lutidine/THF (1:1:8), and N-methyl imidazole/THF is added tocap the unreacted 5'-hydroxyl group. The product is washed withacetonitrile.

The carrier containing the compound is treated with 30% aqueous ammoniumhydroxide solution for 90 minutes and then incubated at 55° C. for 12hours. The aqueous solution is filtered, concentrated under reducedpressure and then treated at room temperature with 1.0M solution oftetra-n-butyl ammonium fluoride in THF to give a phosphorothioate dimerof T-T.

B. Synthesis of C-T phosphorothioate dimer

100 milligram (4 mmole) of 5'-O-Dimethoxytritylthymidine bonded to CPG(controlled pore glass) through an ester linkage is taken in a glassreactor, and a dichloromethane solution of 2% dichloroacetic acid(volume/volume) is added to deprotect the 5'-hydroxyl group. The productis washed with acetonitrile. Then, a 0.2M solution ofN4-Benzoyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxycytidine-3'-O-(1,1,1-triphenyl-1-sila-2-butenyl-N,N-diisopropylphosphoramidite)in acetonitrile and a 0.4M solution of 1H-tetrazole in acetonitrile isadded, and reacted at room temperature for 5 minutes. The product iswashed with acetonitrile, and then a 0.05M solution of Beaucage reagentin acetonitrile is added and reacted at room temperature for 5 minutes.This sulfurization step is repeated one more time for 5 minutes. Thesupport is washed with acetonitrile and then a solution of aceticanhydride/lutidine/THF (1:1:8), and N-methyl imidazole/THF is added tocap the unreacted 5'-hydroxyl group. The product is washed withacetonitrile.

The carrier containing the compound is treated with 30% aqueous ammoniumhydroxide solution for 90 minutes and then incubated at 55° C. for 12hours. The aqueous solution is filtered, concentrated under reducedpressure and then treated at room temperature with 1.0M solution oftetra-n-butyl ammonium fluoride in THF to give a phosphorothioate dimerof dC-T.

D. Synthesis of 5'-TTTTTTT-3' phosphorothioate heptamer

50 milligram (2 mole) of 5'-O-Dimethoxytritylthymidine bonded to CPG(controlled pore glass) through an ester linkage is taken in a glassreactor, and a dichloromethane solution of 2% dichloroacetic acid(volume/volume) is added to deprotect the 5'-hydroxyl group. The productis washed with acetonitrile. Then, a 0.2M solution of5'-O-(4,4'-dimethoxytrityl)thymidine-3'-O-(1,1,1-triphenyl-1-sila-2-butenyl-N,N-diisopropylphosphoramidite)in acetonitrile and a 0.4M solution of 1H-tetrazole in acetonitrile isadded, and reacted at room temperature for 5 minutes. The product iswashed with acetonitrile, and then a 0.05M solution of Beaucage reagentin acetonitrile is added and reacted at room temperature for 5 minutes.This sulfurization step is repeated one more time for 5 minutes. Thesupport is washed with acetonitrile and then a solution of aceticanhydride lutidine/THF (1:1:8), and N-methyl imidazole/THF is added tocap the unreacted 5'-hydroxyl group. The product is washed withacetonitrile.

This complete cycle is repeated five more times to get the completelyprotected thymidine heptamer. The carrier containing the compound istreated with 30% aqueous ammonium hydroxide solution for 90 minutes atroom temperature. The aqueous solution is filtered, concentrated underreduced pressure to give a phosphorothioate heptamer of TTTTTTT.

E. Synthesis of 5'-d(GACT)-3' phosphorothioate tetramer

50 milligram (2 mmole) of 5'-O-dimethoxytritylthymidine bonded to CPG(controlled pore glass) through an ester linkage is taken in a glassreactor, and a dichloromethane solution of 2% dichloroacetic acid(volume/volume) is added to deprotect the 5'-hydroxyl group. The productis washed with acetonitrile. Then, a 0.2M solution of5'-O-(4,4'-dimethoxytrityl)thymidine-3'-O-(1,1,1-triphenyl-1-sila-2-butenyl-N,N-diisopropylphosphoramidite)in acetonitrile and a 0.4M solution of 1H-tetrazole in acetonitrile isadded, and reacted at room temperature for 5 minutes. The product iswashed with acetonitrile, and then a 0.05M solution of Beaucage reagentin acetonitrile is added and reacted at room temperature for 5 minutes.This sulfurization step is repeated one more time for 5 minutes. Thesupport is washed with acetonitrile and then a solution of aceticanhydride/lutidine/THF (1:1:8), and N-methyl imidazole/THF is added tocap the unreacted 5'-hydroxyl group. The product is washed withacetonitrile.

A dichloromethane solution of 2% dichloroacetic acid (volume/volume) isadded to deprotect the 5'-hydroxyl group. The product is washed withacetonitrile. Then, a 0.2M solution ofN4-benzoyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxycytidine-3'-O-(1,1,1-triphenyl-1-sila-2-butenyl-N,N-diisopropylphosphoramidite)in acetonitrile and a 0.4M solution of 1H-tetrazole in acetonitrile isadded, and reacted at room temperature for 5 minutes. The product iswashed with acetonitrile, and then a 0.05M solution of Beaucage reagentin acetonitrile is added and reacted at room temperature for 5 minutes.This sulfurization step is repeated one more time for 5 minutes. Thesupport is washed with acetonitrile and then a solution of aceticanhydride/lutidine/THF (1:1:8), and N-methyl imidazole/THF is added tocap the unreacted 5'-hydroxyl group. The product is washed withacetonitrile.

A dichloromethane solution of 2% dichloroacetic acid (volume/volume) isadded to deprotect the 5'-hydroxyl group. The product is washed withacetonitrile. Then, a 0.2M solution ofN6-benzoyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxyadenosine-3'-O-(1,1,1-triphenyl-1-sila-2-butenyl-N,N-diisopropylphosphoramidite)in anhydrous acetonitrile and a 0.4M solution of 1H-tetrazole inacetonitrile is added, and reacted at room temperature for 5 minutes.The product is washed with acetonitrile, and then a 0.05M solution ofBeaucage reagent in acetonitrile is added and reacted at roomtemperature for 5 minutes. This sulfurization step is repeated one moretime for 5 minutes. The support is washed with acetonitrile and then asolution of acetic anhydride/lutidine/THF (1:1:8), and N-methylimidazole/THF is added to cap the unreacted 5'-hydroxyl group. Theproduct is washed with acetonitrile.

A dichloromethane solution of 2% dichloroacetic acid (volume/volume) isadded to deprotect the 5'-hydroxyl group. The product is washed withacetonitrile. Then, a 0.2M solution ofN2-isobutyryl-5'-O-(4,4'-dimethoxytrityl)2'-deoxyguanosine-3'-O-(1,1,1-triphenyl-1-sila-2-butenyl-N,N-diisopropylphosphoramidite)in acetonitrile and a 0.4M solution of 1H-tetrazole in acetonitrile isadded, and reacted at room temperature for 5 minutes. The product iswashed with acetonitrile, and then a 0.05M solution of Beaucage reagentin acetonitrile is added and reacted at room temperature for 5 minutes.This sulfurization step is repeated one more time for 5 minutes. Thesupport is washed with acetonitrile and then a solution of aceticanhydride/lutidine/THF (1:1:8), and N-methyl imidazole/THF is added tocap the unreacted 5'-hydroxyl group. The product is washed withacetonitrile.

The carrier containing the compound is treated with 30% aqueous ammoniumhydroxide solution for 90 minutes at room temperature and then incubatedat 55° C. for 24 hours. The aqueous solution is filtered, concentratedunder reduced pressure to give a phosphorothioate tetramer of5'-dG-dA-dC-T-3'.

EXAMPLE 19

3'-O-Levulinylthymidine

The title compound was synthesized according a published procedure, seeG. Kumar, M. S. Poonian, J. Org. Chem. 1984, 49, 4905-4912.

EXAMPLE 20

3'-O-Levulinyl--N2-isobutyryl-2'-deoxyguanosine

5'-DMT-N2-isobutyryl-2'-deoxyguanosine (71.6 g, 0.112 mol) istransferred to a 1000 ml flask and anhydrous dioxane (700 ml) is addedand stirred until the solution becomes homogeneous. Thendicyclohexylcarbodiimide (57.8 g, 0.280 mol), levulinic acid (25.9 g,0.224 mol) and 4-dimethylaminopyridine (0.56 g) are added and vigorouslystirred using magnetic stirring. After 3 hours, the reaction mixture isfiltered, the solid residue washed with ethyl acetate (250 ml). Thefilterates are combined and concentrated to afford a product.

This product is dissolved in dichloromethane (400 ml) and 2.5% DCA indichloromethane (160 ml) is added and stirred. After 1 hour, thereaction mixture is diluted with dichloromethane (400 ml) and washedwith saturated sodium bicarbonate solution. The organic layer isseparated, dried and concentrated. The crude product is purified byflash chromatography using silica gel to give the title compound.

EXAMPLE 21

Synthesis of T-T phosphorothioate dimer

To a stirred solution of 3'-O-levulinylthymidine (5 mole) and1H-tetrazole (5 mole) in anhydrous acetonitrile (25 ml) at roomtemperature under argon is added a solution of5'-O-(4,4'-dimethoxytrityl)thymidine-3'-O-(4-diphenylmethylsilyl-2-butenylN,N-diisopropyl phosphoramidite) (6 mole) in acetonitrile (20 ml). Afterstirring for 3 h, the sulfurizing reagent (a mixture of sulfur (200mmole)/triethylamine (20 mole) in dichloromethane (75 ml) is added allat once. After 5 hours, the reaction mixture is filtered andconcentrated. The crude product is purified by flash chromatographyusing silica gel and ethylacetate/hexane as eluents.

EXAMPLE 22

Deprotection of 3'-O-levulinyl group

5'-(O-4,4'-Dimethoxytrityl)-3'-(O-levulinyl)-thymidine dimer (35.0 g) isdissolved in an ice-cold solution of hydrazine-hydrate (10.0 g),pyridine (240 ml) and acetic acid (240 ml). After 10 minutes, ice isadded, followed by extraction with dichloromethane. The organic phase isdried over sodium sulfate, filtered and the solvent is removed. Theresidue is purified by silica gel column chromatography (ethylacetate/n-hexanes 1:1, then ethyl acetate, 0.1% triethylamine) to affordthe desired product.

EXAMPLE 23

Synthesis of thymidyl-thymidine dimer amidite

Under argon, a solution of 1H tetrazole (5 mmol) and4-cyano-2-butenyl-N,N,N',N'-tetraisopropylphosphorodiamidite (30 mmol)in dry acetonitrile (100 ml) is added to thymidyl-thymidine dimer (20mmol). After 2 hours, ethyl acetate is added and the solution isextracted with aqueous sodium bicarbonate. The organic phase is driedover sodium sulfate and the solvent is removed under reduced pressure.The residue is purified by column chromatography to afford the desiredproduct.

EXAMPLE 24

Synthesis of C-T-phosphorothioate dimer

To a stirred solution of 3'-O-levulinylthymidine (5 mmole) and1H-tetrazole (5 mmole) in anhydrous acetonitrile (25 ml) at roomtemperature under argon is added a solution of5'-O-(4,4'-dimethoxytrityl)-2'-deoxycytidine-3'-O-(4-diphenylmethylsilyl-2-butenylN,N-diisopropyl phosphoramidite) (6 mmole) in acetonitrile (20 ml).After stirring for 3 h, the sulfurizing reagent (a mixture of sulfur(200 mmole)/triethylamine (20 mole) in dichloromethane (75 ml) is addedall at once. After 5 hours, the reaction mixture is filtered andconcentrated. The crude product is purified by flash chromatographyusing silica gel and ethylacetate/hexane as eluents.

EXAMPLE 25

Deprotection of 3'-O-levulinyl group

5'-(O-4,4'-Dimethoxytrityl)-3'-(O-levulinyl)-2'-deoxycytidinyl-thymidinedimer (35.0 g) is dissolved in an ice-cold solution of hydrazine-hydrate(10.0 g), pyridine (240 ml) and acetic acid (240 ml). After 10 minutes,ice is added, followed by extraction with dichloromethane. The organicphase is dried over sodium sulfate, filtered and the solvent is removed.The residue is purified by silica gel column chromatography (ethylacetate/n-hexanes 1:1, then ethyl acetate, 0.1% triethylamine) to affordthe desired product.

EXAMPLE 26

Synthesis of 2'-deoxycytidinyl-thymidine dimer amidite

Under argon, a solution of 1H tetrazole (5 mmol) and4-cyano-2-butenyl-N,N,N',N'-tetraisopropylphosphorodiamidite (30 mmol)in dry acetonitrile (100 ml) is added to 2'-deoxycytidinyl-thymidinedimer (20 mmol). After 2 hours, ethyl acetate is added and the solutionis extracted with aqueous sodium bicarbonate. The organic phase is driedover sodium sulfate and the solvent is removed under reduced pressure.The residue is purified by column chromatography to afford the desiredproduct.

EXAMPLE 27

Synthesis of d(A-G)-phosphorothioate dimer

To a stirred solution of 3'-O-levulinyl-2'-deoxyguanosine (5 mole) and1H-tetrazole (5 mole) in anhydrous acetonitrile (25 ml) at roomtemperature under argon is added a solution of5'-O-(4,4'-dimethoxytrityl)-2'-deoxyadenosine-3'-O-(4-diphenylmethylsilyl-2-butenylN,N-diisopropyl phosphoramidite) (6 mole) in acetonitrile (20 ml). Afterstirring for 3 h, the sulfurizing reagent (a mixture of sulfur (200mmole)/triethylamine (20 mmole) in dichloromethane (75 ml) is added allat once. After 5 hours, the reaction mixture is filtered andconcentrated. The crude product is purified by flash chromatographyusing silica gel and ethyl acetate/hexane as eluents.

EXAMPLE 28

Deprotection of 3'-O-levulinyl group

5'-(O-4,4'-Dimethoxytrityl)-3'-(O-levulinyl)-2'-deoxyadenosinyl-2'-deoxyguanosine dimer (35 g) is dissolved in an ice-coldsolution of hydrazine-hydrate (10.0 g), pyridine (240 ml) and aceticacid (240 ml). After 10 minutes, ice is added, followed by extractionwith dichloromethane. The organic phase is dried over sodium sulfate,filtered and the solvent is removed. The residue is purified by silicagel column chromatography (ethyl acetate/n-hexanes 1:1, then ethylacetate, 0.1% triethylamine) to afford the desired product.

EXAMPLE 29

Synthesis of 2'-deoxyadenosinyl-2'-deoxyguanosine dimer amidite

Under argon, a solution of 1H tetrazole (5 mmol) and4-cyano-2-butenyl-N,N,N',N'-tetraisopropylphosphorodiamidite (30 mmol)in dry acetonitrile (100 ml) is added to2'-deoxyadenosinyl-2'-deoxyguanosine dimer (20 mmol). After 2 hours,ethyl acetate is added and the solution is extracted with aqueous sodiumbicarbonate. The organic phase is dried over sodium sulfate and thesolvent is removed under reduced pressure. The residue is purified bycolumn chromatography to afford the desired product.

EXAMPLE 30

Synthesis of 5'-TTTTTTT-3' phosphorothioate heptamer

50 milligram (2 mmole) of 5'-O-dimethoxytritylthymidine bonded to CPG(controlled pore glass) through an ester linkage is taken in a glassreactor, and a dichloromethane solution of 2% dichloroacetic acid(volume/volume) is added to deprotect the 5'-hydroxyl group. The productis washed with acetonitrile. Then, a 0.2M solution of5'-O-(4,4'-dimethoxytrityl)-thymidyl-thymidine-3'-O-(4-cyano-2-butenylN,N-diisopropylphosphoramidite) in acetonitrile and a 0.4M solution of1H-tetrazole in acetonitrile is added, and reacted at room temperaturefor 5 minutes. The product is washed with acetonitrile, and then a 0.05Msolution of Beaucage reagent in acetonitrile is added and reacted atroom temperature for 5 minutes. This sulfurization step is repeated onemore time for 5 minutes. The support is washed with acetonitrile andthen a solution of acetic anhydride/lutidine/THF (1:1:8), and N-methylimidazole/THF is added to cap the unreacted 5'-hydroxyl group. Theproduct is washed with acetonitrile.

This complete cycle is repeated two more times to get the completelyprotected thymidine heptamer. The carrier containing the compound istreated with 30% aqueous ammonium hydroxide solution for 90 minutes atroom temperature and then incubated at 55° C. for 1 hour. The aqueoussolution is filtered, concentrated under reduced pressure to give aphosphorothioate heptamer of 5'-TTTTTTT-3'.

EXAMPLE 31

Synthesis of 5'-d(TTCTAGT)-3' phosphorothioate heptamer

50 milligram (2 mmole) of 5'-O-dimethoxytritylthymidine bonded to CPG(controlled pore glass) through an ester linkage is taken in a glassreactor, and a dichloromethane solution of 2% dichloroacetic acid(volume/volume) is added to deprotect the 5'-hydroxyl group. The productis washed with acetonitrile. Then, a 0.2M solution of5'-O-(4,4'-dimethoxytrityl)-2'-deoxyadenosinyl-2'-deoxyguanosine-3'-O-(4-cyano-2-butenylN,N-diisopropylphosphoramidite) in acetonitrile and a 0.4M solution of1H-tetrazole in acetonitrile is added, and reacted at room temperaturefor 5 minutes. The product is washed with acetonitrile, and then a 0.05Msolution of Beaucage reagent in acetonitrile is added and reacted atroom temperature for 5 minutes. This sulfurization step is repeated onemore time for 5 minutes. The support is washed with acetonitrile andthen a solution of acetic anhydride/lutidine/THF (1:1:8), and N-methylimidazole/THF is added to cap the unreacted 5'-hydroxyl group. Theproduct is washed with acetonitrile.

A dichloromethane solution of 2% dichloroacetic acid (volume/volume) isadded to deprotect the 5'-hydroxyl group. The product is washed withacetonitrile. Then, a 0.2M solution of5'-O-(4,4'-dimethoxytrityl)-2'-deoxycytidinyl-thymidine-3'-O-(4-cyano-2-butenylN,N-diisopropylphosphoramidite) in acetonitrile and a 0.4M solution of1H-tetrazole in acetonitrile is added, and reacted at room temperaturefor 5 minutes. The product is washed with acetonitrile, and then a 0.05Msolution of Beaucage reagent in acetonitrile is added and reacted atroom temperature for 5 minutes. This sulfurization step is repeated onemore time for 5 minutes. The support is washed with acetonitrile andthen a solution of acetic anhydride/lutidine/THF (1:1:8), and N-methylimidazole/THF is added to cap the unreacted 5'-hydroxyl group. Theproduct is washed with acetonitrile.

A dichloromethane solution of 2% dichloroacetic acid (volume/volume) isadded to deprotect the 5'-hydroxyl group. The product is washed withacetonitrile. Then, a 0.2M solution of5'-O-(4,4'-dimethoxytrityl)-thymidyl-thymidine-3'-O-(4-cyano-2-butenylN,N-diisopropylphosphoramidite) in anhydrous acetonitrile and a 0.4Msolution of 1H-tetrazole in acetonitrile is added, and reacted at roomtemperature for 5 minutes. The product is washed with acetonitrile, andthen a 0.05M solution of Beaucage reagent in acetonitrile is added andreacted at room temperature for 5 minutes. This sulfurization step isrepeated one more time for 5 minutes. The support is washed withacetonitrile and then a solution of acetic anhydride/lutidine/THF(1:1:8), and N-methyl imidazole/THF is added to cap the unreacted5'-hydroxyl group. The product is washed with acetonitrile.

The carrier containing the compound is treated with 30% aqueous ammoniumhydroxide solution for 90 minutes at room temperature and then incubatedat 55° C. for 24 hour. The aqueous solution is filtered, concentratedunder reduced pressure to give a phosphorothioate heptamer of5'-d(TTCTAGT)-3'.

It is intended that each of the patents, publications, and otherpublished documents mentioned or referred to in this specification beherein incorporated by reference in its entirety.

Those skilled in the art will appreciate that numerous changes andmodifications may be made to the preferred embodiments of the inventionand that such changes and modifications may be made without departingfrom the spirit of the invention. It is therefore intended that theappended claims cover all such equivalent variations as fall within thetrue spirit and scope of the invention.

What is claimed is:
 1. A compound having the Formula VII:

    A--X.sub.1 --CH.sub.2 --CH═CH--CH.sub.2 --Z            VII

wherein: X₁ is O or S; A is (R₇) (R₈)P--; R₈ is R₅, or has the FormulaX: ##STR17## wherein: each R₁, is, independently, H, --OH, --F, --O--X₃--D; X₃ is alkyl having from 1 to 10 carbons; D is H, amino, protectedamino, alkyl substituted amino, imidazole, or (--O--X₃)_(p), where p is1 to about 10; each X₂ is O or S; R₅ is --N(R₆)₂, or a heterocycloalkylor heterocycloalkenyl ring containing from 4 to 7 atoms, and having upto 3 heteroatoms selected from the group consisting of nitrogen, sulfur,and oxygen; each Q is --X₁ H or --X₁ --CH₂ --CH═CH--CH₂ --Z; m is 0 toabout 50; each B, independently, is a naturally occurring ornon-naturally occurring nucleobase or a protected naturally occurring ornon-naturally occurring nucleobase; and R₇ is R₅, or has the FormulaVIII: ##STR18## wherein: R₃ is hydrogen, a hydroxyl protecting group, ora linker connected to a solid support; and n is 0 to about 50; with theproviso that the sum of m and n do not exceed 50; and Z is CN, halogen,NO₂, alkaryl, sulfoxyl, sulfonyl, thio, substituted sulfoxyl,substituted sulfonyl, or substituted thio, wherein the substituents areselected from the group consisting of alkyl, aryl, or alkaryl.
 2. Thecompound of claim 1 wherein Z is CN.
 3. The compound of claim 2 whereinX₁ is O.
 4. The compound of claim 2 wherein X₁ is S.
 5. The compound ofclaim 2 wherein A is H.
 6. The compound of claim 2 wherein A is--P(R₅)₂.
 7. The compound of claim 5 wherein R₅ is --N(CH(CH₃)₂)₂. 8.The compound of claim 1 wherein R₇ has the Formula VIII.
 9. The compoundof claim 8 wherein n is 1 to
 30. 10. The compound of claim 8 wherein nis 1 to about
 25. 11. The compound of claim 8 wherein n is
 0. 12. Thecompound of claim 2 wherein Z is CN; X₁ is O; and A is H.
 13. Thecompound of claim 2 wherein Z is CN; X₁ is S; and A is H.
 14. Thecompound of claim 2 wherein Z is CN; X₁ is O; and each R₆ is isopropyl.15. The compound of claim 2 wherein Z is CN; X₁ is S; and each R₆ isisopropyl.
 16. The compound of claim 1 having the Formula IV: ##STR19##17. The compound of claim 16 wherein R₂ is a linker connected to a solidsupport.
 18. The compound of claim 16 wherein R₂ is hydrogen.
 19. Thecompound of claim 16 wherein m and n are each
 0. 20. The compound ofclaim 16 wherein Z is CN; and X₁ is O.
 21. A compound having theFormula: ##STR20## wherein: each R₁, is, independently, H, --OH, --F, or--O--X₃ --D;D is H, amino, protected amino, alkyl substituted amino,imidazole, or (--O--X₃)_(p), where p is 1 to about 10; R₂ is a hydroxylprotecting group, or a linker connected to a solid support; R₃ ishydrogen, a hydroxyl protecting group, or a linker connected to a solidsupport, provided that R₂ and R₃ are not both simultaneously a linkerconnected to a solid support; X₁ is O or S; each X₂ is O or S; each X₃is alkyl having from 1 to 10 carbons; each Q is --X₁ H or --X₁ --CH₂--CH═CH--CH₂ --Z; m and n are each independently an integer from 0 toabout 50, provided that the sum of m and n does not exceed 50; each B,independently, is a naturally occurring or non-naturally occurringnucleobase or a protected naturally occurring or non-naturally occurringnucleobase; and Z is CN, halogen, NO₂, alkaryl, sulfoxyl, sulfonyl,thio, substituted sulfoxyl, substituted sulfonyl, or substituted thio,wherein the substituents are selected from the group consisting ofalkyl, aryl, or alkaryl.
 22. The compound of claim 21 wherein R₂ is alinker connected to a solid support.
 23. The compound of claim 21wherein R₂ is hydrogen.
 24. The compound of claim 21 wherein m and n areeach
 0. 25. The compound of claim 21 wherein Z is CN; and X₁ is O. 26.The compound of claim 8 wherein R₈ is R₅.
 27. The compound of claim 26wherein n is from 2 to 50, and each Q has the Formula --X₁ --CH₂--CH═CH--CH₂ --Z.
 28. An oligomeric compound comprising a moiety havingthe Formula IX; ##STR21## wherein: Z is CN, halogen, NO₂, alkaryl,sulfoxyl, sulfonyl, thio, substituted sulfoxyl, substituted sulfonyl, orsubstituted thio, wherein the substituents are selected from the groupconsisting of alkyl, aryl, or alkaryl; andX₁ is O or S;prepared by thesteps of: (a) providing a compound having the Formula II: ##STR22##wherein each R₁, is, independently, H, --OH, --F, or --O--X₃ --D; X₃ isalkyl having from 1 to 10 carbons; D is H, amino, protected amino, alkylsubstituted amino, imidazole, or (--O--X₃)_(p), where p is 1 to about10; each X₂ is O or S; R₃ is hydrogen, a hydroxyl protecting group, or alinker connected to a solid support: each B, independently, is anaturally occurring or non-naturally occurring nucleobase or a protectednaturally occurring or non-naturally occurring nucleobase; n is 0 toabout 50; each Q is --X₁ H or --X₁ --CH₂ --CH═CH--CH₂ --Z; R₅ is--N(R₆)₃, or a heterocycloalkyl or heterocycloalkenyl ring containingfrom 4 to 7 atoms, and having up to 3 heteroatoms selected from thegroup consisting of nitrogen, sulfur, and oxygen; R₆ is straight orbranched chain alkyl having from 1 to 10 carbons;(b) reacting thecompound of Formula II with a compound having the Formula III: ##STR23##wherein R_(3a) is hydrogen; and R₂ is a hydroxyl protecting group, or alinker connected to a solid support; (c) oxidizing the product of step(b) to form a further compound having the Formula III, wherein R_(3a) ishydrogen, a hydroxyl protecting group, or a linker connected to a solidsupport.